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Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae)
Arthropod Structure & Development xxx (2014) 1e10
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Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae) Tingting Wang, Liuxing Pan, Yalin Zhang, Wu Dai* Key Laboratory of Plant Protection Resources and Pest Management of the Ministry of Education, Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 August 2014 Accepted 5 December 2014 Available online xxx
Mouthparts associated with feeding behavior and feeding habits are important sensory and feeding structures in insects. To obtain a better understanding of feeding in Cercopoidea, the morphology of mouthparts of the spittlebug, Philagra albinotata Uhler was examined using scanning electron microscopy. The mouthparts of P. albinotata are of the typical piercingesucking type found in Hemiptera, comprising a cone-shaped labrum, a tube-like, three-segmented labium with a deep groove on the anterior side, and a stylet fascicle consisting of two mandibular and two maxillary stylets. The mandibles consist of a dorsal smooth region and a ventral serrate region near the apical half of the external convex region, and bear five nodules or teeth on the dorsal external convex region on the distal extremity; these are regarded as unique features that distinguish spittlebugs from other groups of Hemiptera. The externally smooth maxillary stylets, interlocked to form a larger food canal and a smaller salivary canal, are asymmetrical only in the internal position of longitudinal carinae and grooves. One dendritic canal is found in each maxilla and one in each mandible. Two types of sensilla trichodea, three types of sensilla basiconica and groups of multi-peg structures occur in different locations on the labium, specifically the labial tip with two lateral lobes divided into anterior sensory fields with ten small peg sensilla arranged in a 5 þ 4 þ 1 pattern and one big peg sensillum, and posterior sensory fields with four sensilla trichodea. Compared with those of previously studied Auchenorrhyncha, the mouthparts of P. albinotata may be distinguished by the shape of the mandibles, the multi-peg structures and a tooth between the salivary canal and the food canal on the extreme end of the stylets. The mouthpart morphology is illustrated using scanning electron micrographs, and the taxonomic and putative functional significance of the different structures is briefly discussed. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Hemiptera Philagra albinotata Mouthparts Sentilla Morphology
1. Introduction Cercopoidea, the second most species-rich superfamily in the Cicadomorpha, comprises approximately 3000 described species. The superfamily has been classified into five families: Cercopidae, Aphrophoridae, Clastopteridae, Machaerotidae and Epipygidae (Cryan and Svenson, 2010). Adults feed on the leaves and stems of a variety of plants by inserting their maxillary stylets into the xylem elements and suck the sap (Leopold et al., 2003). The saliva of some species, such as the sugarcane spittlebug, Mahanarva fimbriolata, contains toxic enzymes and feeding by these insects on plants may
* Corresponding author. Tel.: þ86 29 87092509; fax: þ86 29 87092190. E-mail address: [email protected] (W. Dai).
result in the blockage of conducting channels causing dessication of the leaves (Nunes and Camargo-Mathias, 2006). Mouthparts are important sensory and feeding structures in insects and their structure is closely associated with feeding behavior and feeding habits (Ma et al., 2013). Understanding the structure of mouthparts and accompanying sensilla is relevant, not only in host plant selection studies, but also in the study of feeding behavior and sensory physiology (Rani and Madhavendra, 2005). In addition, because mouthpart structures may be characteristic of all members of a genus, family or order of insects, knowledge of mouthpart morphology is useful for classification and identification (Gullan and Cranston, 2005). Previous work on the ultrastructural morphology of Hemiptera mouthparts, using light and scanning electron microscopy, has mainly focused on Heteroptera (Anderson et al., 2006; Boyd, 2003; Cobben, 1978), Aphidoidea (Forbes, 1969;
http://dx.doi.org/10.1016/j.asd.2014.12.001 1467-8039/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001
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Pollard, 1973; Razaq et al., 2000), Psyllidae (Garzo et al., 2012; Liang et al., 2013) and Aleyrodidae (Rosell et al., 1995; Walker and Gordh, 1989) of Sternorrhyncha, and Cicadellidae (Tavella and Arzone, 1993; Leopold et al., 2003; Wiesenborn, 2004; Zhao et al., 2010; Ammar and Hall, 2012) and Fulgoroidea (Brentassi and de Remes Lenicov, 2007; Dai et al., 2014; Liang, 2005) of Auchenorrhyncha. Apart from some brief notes on the stylets of Philaenus spumarius (L.) (Pollard, 1971) and Notozulia entreriana (Berg) (Paladini et al., 2008), and the interlocking pattern of maxillae and mandibles of n) (Cobben, 1978), fine structure of the Aphrophora alni (Falle mouthparts in spittlebugs has received little attention. Information on the distribution of the sensilla on the mouthparts, the relationship between mouthpart structure and function in feeding and the utility of this information for classification of spittlebugs is not available. In the present work, the mouthpart morphology of Philagra albinotata Uhler is described to provide comparative data elucidating variation within the family, contribute to knowledge of feeding behavior, and also to reveal characters that may be useful for future studies in taxonomy and phylogeny of Auchenorrhyncha, especially within Cercopoidea. 2. Material and methods 2.1. The insect Adult P. albinotata were collected at Huoditang in Ningshan, Shaanxi Province, China in July 2011, using a sweep net and preserved in 75% ethanol. Voucher specimens were deposited in the Entomological Museum of Northwest A&F University, Yangling, Shaanxi Province, China. 2.2. Sample preparation for SEM The heads of randomly selected five female and male specimens respectively were freed from the body. The mouthparts were excised and dissected using fine dissecting needles under 40 magnification (Nikon SMZ 1500, stereomicroscope, Japan). Specimens were then fixed with 2.5% glutaraldehyde for 12 h at 4 C, followed by washing in phosphate buffered saline (PBS, 0.1 M, pH7.2) for 15 min four times and cleaned in an ultrasonic cleaner for 15 s three times before dehydration in a graded series of 30%, 50%, 70%, 80%, 90% and 95% ethanol for 20 min each and 100% ethanol for 30 min twice. Specimens then underwent replacement in the mixture of 100% alcohol and 100% tertiary butyl alcohol mixed in sequential ratios (3:1, 1:1 and 1:3 by volume) for 15 min each time. Samples were then soaked in 100% tert-Butanol for 30 min before being placed into a freeze-drier with liquid CO2 for 3 h. Thereafter, the samples were mounted on aluminum stubs with double-sided copper sticky tape and sputtered with gold/ palladium (40/60) in a LADD SC-502 (Vermont, USA) high resolution sputter coater. The samples were subsequently examined with a Hitachi S-3400N scanning electron microscope operated at 15 kV (Zhao et al., 2010). All measurements are given as mean ± S.E. 3. Results 3.1. Gross morphology of mouthparts As in other Auchenorrhyncha, the mouthparts of Philagra albinotata arise from the posteroventral part of the head capsule (Fig. 1A) and are typical piercingesucking form (Fig. 1A and B) composed of the labrum (Lm) (Figs. 1B and 2B), a tube-like labium (Lb) (Fig. 1) and a stylet fascicle (Sf) consisting of two mandibular
(Md) and two maxillary stylets (Mx). The three-segmented labium has a longitudinal groove in the middle of the venter, called the labial groove (Lg) (Fig. 1B), which envelops the stylet fascicle comprising two inner maxillary stylets partially surrounded by two shorter and serrate-edged mandibular stylets, and proximally by the small cone-shaped labrum. The surface of the labium has different types of sensilla symmetrically arranged on both sides of the labial groove or positioned on the labial apex (Fig. 1B). 3.2. Labrum The cone-shaped labrum (Lm) is very short (411 ± 6 mm, n ¼ 5) (Fig. 2A and B), and is attached to the anterior margin of the anteclypeus and overlies the 1st and 2nd labial segments (Figs. 1B and 2A). The surface of the labrum is covered with a few scattered short triangular spines (Fig. 2C). Some clusters of pegs on the lateral edge and the terminus are more distinct and palmate (Fig. 2C). 3.3. Labium The labium (Lb) (also known as the rostrum or proboscis) is tubular in shape and composed of three segments (Fig. 1C). The anterior surface of the labium is bisected by a deep labial groove (Lg) (Fig. 1B), extending its entire length, enclosing two mandibular and two maxillary stylets. Sensilla are mainly distributed symmetrically on each side of the labial groove (Lg) and distally, but fewer occur on the dorsal and lateral surface (Fig. 1B and C). The tip of the labial segment is flattened with an opening from which the apices of the stylets are extended (Figs. 1B, C, and 5B). The total length of the labium is 2109 ± 20 mm (n ¼ 5). It is broad and of uniform width through most of its length with the distal segment widening near the tip. The first labial segment is the shortest (567.3 ± 9.6 mm, n ¼ 5) with most of it concealed by the overlapping anteclypeus (Fig. 1B). There are scarcely any sensilla trichodea I on this segment (Fig. 1C). The second labial segment is the longest (781 ± 17 mm, n ¼ 5) of the three segments (Figs. 1C and 2A), with a small round tumid area close to the first segment (Fig. 3A). There are two types of sensilla, sensilla trichodea I and sensilla basiconica I. Most sensilla trichodea I (s.t.I) are arranged symmetrically on each side of the labial groove (Fig. 2A and D) and on the lateral surface, and a few are arranged on posterior surface near the junction of the middle and third segments (Fig. 3A). Sensilla trichodea I, ranging from 75 mm to 140 mm in length, are slender and slightly curved with pointed tips. The surface has longitudinal grooves that spiral slightly around the shaft, and gradually converge on the obverse side, gradually disappearing near the distal part (Fig. 2D and E). Sensilla basiconica I (<30 mm in length) are short, stout basiconic pegs with a blunt tip, with a minute longitudinal groove in the shaft, arise from sunken pits (Fig. 2D), and are randomly distributed on the ventrum and dorsum of the labium. Numerous clusters of short denticles are scattered over the anterior surface of second segment (Figs. 2A and 3A). The third labial segment is 761 ± 5 mm (n ¼ 5) long, tubular, of uniform width from base to apical 1/4 then widens to the apex (Figs. 1C, 3B and D). There are some sensilla trichodea I and several sensilla basiconica I arranged on each side of the labial groove and randomly distributed on the dorsum of the labium (Fig. 3B, D, F and G). Two sensilla basiconica II are present on each side of the junction of the second and third segments (Fig. 3D and E). The labial tip forms two lateral lobes separated by the anterior stylet groove and posterior groove. Each lateral lobe has an anterior sensory field located laterad of the stylet groove and a posterior sensory field located behind the stylet groove (Fig. 4C). Each
Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001
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Fig. 1. Scanning electron micrographs showing the head including the mouthparts. (A) SEM micrograph of the head of adult Philagra albinotata showing the position of the mouthparts in anteroventral view. (B) Anterior view of labrum and labium showing sensilla symmetrically located on each side of the labial groove (Lg). (C) Dorsal view of mouthparts showing three-segmented labium (IeIII). Bars: A ¼ 1 mm; B and C ¼ 500 mm.
anterior sensory field consists of two kinds of short, stout basiconic pegs (Fig. 4C). The larger one is sensilla basiconica III (S.b.III) at the center of each side of the labial opening, with a bulbous base, and measuring 60 mm in length (Fig. 4B and C). This bristle is straight with a relatively blunt tip (Fig. 4B and C). Ten smaller sensilla basiconica II (S.b.II) (Fig. 4C and E) are blunt tipped, 25 mme28 mm in length, and located on either side of the labial groove, arising from a slightly raised flexible socket which tightly embraces the base of the shaft (Fig. 4C and E). The cuticular walls bear longitudinal grooves with a molting pore near the base. The sensilla basiconica II are divided into three groups, numbered 1, 5 and 4 from anterior to posterior, respectively. The posterior sensory field consists of two long tapering sensilla trichodea II (S.t.II) on each side of the posterior groove (Fig. 4A and D). The sensilla trichodea II are 75.5 ± 0.7 mm (n ¼ 8) in length and are symmetrically located on either side near the tip of the labium (Fig. 4A and D). They are straight, tapered setae with sharp apices and arise from a slightly raised flexible socket which tightly embraces the base of the shaft, and its surface bears longitudinal grooves with a cavity near the base (Fig. 4D). There are multi-peg structures (MS) on the ventral margin of stylet opening of the labial tip (Figs. 3B, C, 4A and C).
These structures include numerous tall (tp) and short (sp) pegs, grouped on the ventral edge of the opening of the labial tip (Fig. 3C). The short pegs are mostly located on the outer edge of the cluster, away from the labial tip (Fig. 3C). The pegs of these structures are directed towards the end of the terminal segment. 3.4. Stylet fascicle The needle-like stylet fascicle (Sf) comprising two mandibular (Md) and two maxillary stylets (Mx) (Fig. 5A) is housed in the labium and protrudes from the flattened labial tip (Fig. 5B). The mandibular (Md) stylets are crescent-shaped in cross section, located laterad of each maxillary stylets and are slightly shorter than the maxillary stylets (Fig. 5B). The inner surface of the mandibular stylet is smooth (Fig. 5E), to facilitate free movement of the maxillary stylets when probing the plant tissue. On the dorsal margin of the convex external surface of each stylet tip are 4e6 nodules (Fig. 5C and D), which are among the most noticeable features of the mandibular stylet. The ventral margin of each mandibular stylet is serrate, with a corresponding row of obliquely transverse ridges, ranging from 5.7 mm to 7.2 mm (Fig. 5C, D and F),
Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001
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Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001
T. Wang et al. / Arthropod Structure & Development xxx (2014) 1e10
which are prominent at the tip of the stylet and gradually reduced in a step-wise fashion toward the base. The cross-section of the stylet fascicle shows that each mandibular stylet has a dendritic canal (Fig. 5G), a large duct that runs the length of the stylet and is located centrally in the thickest portion of each structure (Fig. 5G). In contrast to the mandibles, the maxillary stylets are not bilaterally symmetrical but are more complex (Fig. 5B). The external surface of the maxillary stylets is smooth (Fig. 5H). The two stylets are held together by internal longitudinal grooves and their interlocking mechanism. The two internal longitudinal grooves interlock with each other forming a food canal (Fc) which is used to suck plant juice, and a salivary canal (Sc) that delivers saliva to the plant (Fig. 5G and I). The two maxillary stylets in cross-section form approximately an ellipse due to the joining of the two crescentshaped sides. The hollow food canal is approximately circular in cross section and located in the center. The salivary canal (Sc) is also circular and located laterally on the inner side of one of the joints where the stylets interlock (Fig. 5G and I). The diameter of the food canal is 30.8 ± 0.5 mm (n ¼ 4), which is much larger than the salivary canal of 8.0 ± 0.2 mm (n ¼ 4) in diameter. On the extreme end of the stylets, the salivary canal has a controllable opening which is adjusted by a tooth and connects with the food canal through which it may deliver a small portion of secreted saliva to digest plant juices or sap (Fig. 5I). Within each maxillary stylet there is one approximately semicircular dendritic canal, smaller than the food canal, but larger than the salivary and dendritic canals in the mandibular stylet (Fig. 5G). These canals are distinct from the food and salivary canals. 4. Discussion We provide the first description of the fine structure of the mouthparts of the spittlebug. There are a number of morphological similarities in the mouthparts of this insect to those of Auchenorrhyncha species described previously (Anderson et al., 2006; Boyd, 2003; Forbes, 1969; Pollard, 1973; Garzo et al., 2012; Liang et al., 2013; Rosell et al., 1995; Zhao et al., 2010). The basic structure of the mouthparts consists of a cone-shaped labrum covered with some triangular spines, a cylindrical, three-segmented labium with abundant different types of sensilla mainly symmetrically distributed on each side of the labial groove, a needle-like stylet fascicle comprising two mandibular and two maxillary stylets, a crescentshaped mandibular stylet with several serrate ridges (Sr) on the surface of each stylet tip and one row of numerous tooth-like projections on the lateral edge of each stylet and two maxillary stylets held together forming a food canal (Fc) and a salivary canal (Sc). The basic similarities in the piercingesucking mouthparts of Auchenorrhyncha are likely due to the similarity in their feeding habits (Zhao et al., 2010). Previous study of the fine structure and function of the labrum has apparently been limited to two leafhopper species, Psammotettix striatus (L.) and Homalodisca vitripennis (as Homalodisca coagulata), and one planthopper species, Sogatella furcifera (Dai et al., 2014; Leopold et al., 2003; Zhao et al., 2010). Although there are some common features among hemipteran insects, the shape, size, and density and distribution of the triangular spines on the surface are clearly different in the species studied. The surface of the labrum in Sogatella furcifera (Hemiptera: Delphacidae) is covered with numerous small irregular papillae, and its lateral side
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has sparsely distributed sensilla trichodea (Dai et al., 2014). The labrum in Psammotettix striatus (L.) and Homalodisca vitripennis have small triangular cuticular processes and no sensilla on them, while the surface is smooth in Cacopsylla chinensis, and a sensillum basiconicum I is located on each side of the junction of the labrum and anteclypeus (Dai et al., 2014; Leopold et al., 2003; Liang et al., 2013; Zhao et al., 2010). In our research, the surface of the labrum in P. albinotata is covered with scattered short triangular cuticular processes. Based on the characteristics of the labrum, Aphrophoridae are most similar to Cicadellidae. The sharp end and the abundant protrusions of the mandibular stylets are structures specialized to pierce plant tissues while probing and are also present in other hemipteran insects (Forbes, 1969; Ullman and McLean, 1986; Zhao et al., 2010; Garzo et al., 2012). The limited variation observed among Auchenorrhyncha in the shape of the stylets may be due to the strong functional constraints on these structures and reflects the similar phytophagous feeding habits among species within the group. However, some details of the fine structure differ and these require additional comparative study to determine their functional and phylogenetic significance. In P. albinotata, on the medial surface of the tip of each mandibular stylet, there is a row of ridges (Sr) giving the stylet a serrate appearance. These structures presumably are specialized for perforating plant tissues using stylet movement and the extent of their development may be related to the depth of stylet penetration into the host plant (Garzo et al., 2012; Leopold et al., 2003; Liang et al., 2013; Rani and Madhavendra, 2005; Zhao et al., 2010). Such ridges are also found in other hemipteran insects, but differ in number and pattern among taxa. Each mandibular stylet has five teeth in Delphacidae (Dai et al., 2014) and ten teeth are present in Psyllidae (Garzo et al., 2012; Liang et al., 2013; Pollard, 1970; Ullman and McLean, 1986), whereas more than ten ridges may be found on the mandibles of Aphididae (Forbes, 1969) and nine to twenty teeth occur in different species of leafhoppers (Leopold et al., 2003; Zhao et al., 2010). The teeth are sharper in predacious hemipterans than in phytophagous hemipterans (Anderson et al., 2006; Boyd et al., 2002; Boyd, 2003; Garzo et al., 2012). In P. albinotata, except for five dorsal nodules, numerous ridges on the apical half of the stylet are restricted to the ventral part of the external convex region while the dorsal part is smooth. These structures have not been observed in other studied hemipteran taxa. Study of additional spittlebug species, as well as other hemipterans, is needed to determine whether the serrate mandibular stylet is a unique feature of Cercopoidea distinguishing them from other Hemiptera species. Study of other xylem-feeding Auchenorrhyncha such as cicadas is especially needed in order to determine whether this feature is associated with xylem feeding. Maxillary stylets were observed to be asymmetrical only in the internal position of longitudinal carinae and grooves, and not in their apical shape. As in previously studied leafhoppers and planthoppers, the grooves on the maxillary stylets of P. albinotata Uhler form a salivary canal (Sc) and a food canal (Fc). However, a tooth between the salivary canal and the food canal on the extreme end of the stylets is present in P. albinotata but has not been observed in other Auchenorrhyncha. Based on its morphology and position, this tooth may allow for the saliva to enter the food canal. The prominent dendritic canals within the mandibular and maxillary stylets demonstrate that the dual innervation of the fascicle is extensive and, based on previous reports, probably has a
Fig. 2. SEM of Philagra albinotata. (A) Anterior view of labrum and second labial segment. (B) The cone-shaped labrum (Lm). (C) Enlarged view of outlined box of (B) showing triangular spines (black arrow) and clusters of pegs (white arrow). (D) Anterior view of apical half of the second labial segment showing sensilla trichodea I (S.t.I) and sensilla basiconica I (S.b.I). (E) Enlarged view of sensillum trichodeum I (S.t.I) showing longitudinal grooves that spiral slightly around the shaft and gradually converge on the obverse side. Bars: A ¼ 80 mm; B ¼ 60 mm; C ¼ 8 mm; D ¼ 60 mm; E ¼ 25 mm.
Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001
Fig. 3. (A) Dorsal view of second labial segment showing a small round tumid area (black arrow), sensilla trichodea I (S.t.I) and sensilla basiconica I (S.b.I). (B) Dorsal view of the third labial segment showing sensilla trichodea I (S.t.I), sensilla basiconica I (S.b.I) and multi-peg structures (MS). (C) Enlarged view of multi-peg structures (MS), showing tall (tp) and short (sp) pegs. (D) Anterior view of the third labial segment showing sensilla trichodea I (S.t.I) and sensilla basiconica II (S.b.II). (E) Enlarged view of sensilla basiconica II (S.b.II). (F) Enlarged view of sensilla trichodea I (S.t.I) and sensilla basiconica I (S.b.I). (G) Enlarged view of outlined box of (D) showing sensilla trichodea I (S.t.I) and sensilla basiconica I (S.b.I). Bars: A ¼ 100 mm; B ¼ 150 mm; C ¼ 12 mm; D ¼ 160 mm; E ¼ 6 mm; F ¼ 15 mm; G ¼ 60 mm.
Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001
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Fig. 4. SEM of Philagra albinotata labium. (A) Dorsal view of the apex of third labial segment showing sensilla trichodea II (S.t.II). (B) Ventral view of the apex of third labial segment. (C) Labial tip with stylet bundle removed, showing sensilla basiconica II (S.b.II) and sensilla basiconica III (S.b.III) from ventral side above. (D) Enlarged view of sensilla trichodea II (S.t.II). (E) Enlarged view of sensilla basiconica II (S.b.II). Bars: A ¼ B ¼ 60 mm; C ¼ 50 mm; D ¼ 20 mm; E ¼ 18 mm.
Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001
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Fig. 5. SEM of Philagra albinotata stylets. (A) Whole stylet fascicle. (B) Enlarged view of apex of stylet fascicle showing the outer mandibular stylets (Md) are slightly shorter than the maxillary stylets (Mx). (C) Mandibular stylet (Md) showing serrate ridge (Sr) on the convex external surface. (D) Tip of mandibular stylet (Md), showing serrate ridge (Sr) on the convex external surface and nodule (white arrow). (E) Smooth hollow inner surface in mandibular stylet (Md). (F) Enlarged view of serrate ridge (white arrow). (G) Cross-section of stylet fascicle showing mandibular stylets (Md) and maxillary stylets (Mx), the interlock between the maxillary stylets, food canal (Fc), salivary canal (Sc), one dendritic canal in each maxillary stylet (white asterisk) and one dendritic canal in each mandibular stylet (white asterisk). (H) Maxillary stylets (Mx) with smooth external surfaces. (I) One of the maxillary stylets (Mx) showing inner surface, food canal (Fc) and salivary canal (Sc). Bars: A ¼ 300 mm; B ¼ 60um; C ¼ 15 mm; D ¼ 20 mm; E ¼ 30 mm; F ¼ 12 mm; G ¼ 15 mm; H ¼ 30 mm; I ¼ 15 mm.
Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001
T. Wang et al. / Arthropod Structure & Development xxx (2014) 1e10
proprioceptive function (Foster et al., 1983; Leopold et al., 2003; Zhao et al., 2010). The number of dendritic canals in the maxilla and mandible varies in different species. The studies of Backus and McLean (1982), Zhao et al. (2010), and Dai et al. (2014) clearly show that in Auchenorrhyncha 2e5 dendrites are present in each maxilla and 1e3 in each mandible. In P. albinotata, each mandible has one dendritic canal and each maxilla also has one dendritic canal. Three types of sensilla cover the tip of the labium of the spittlebug species: sensilla trichodea II, sensilla basiconica II and sensilla basiconica III. Although the functions of the sensilla on the labium have not been determined, it has been reported for several hemipterans that such rostral sensilla may respond to different stimuli produced by the host plant, and most of them are chemoreceptors and mechanoreceptors (Backus and McLean, 1982; Brozek and Bourgoin, 2013; Foster et al., 1983; Rani and Madhavendra, 2005; Zhao et al., 2010). The sensilla basiconica and the poreless sensilla trichodea come into direct contact with the host plant surface. The sensilla basiconica III in P. albinotata are extremely similar to those of Homalodisca vitripennis, which are regarded as mechanoreceptors (Leopold et al., 2003). Similar apical labial sensilla have been reported in other leafhoppers (Backus and McLean, 1982; Zhao et al., 2010). Ten sensilla basiconica II located in the dorsal margin of the tip of the labium are similar to those of _ Fulgoromorpha (Foster et al., 1983; Backus, 1985; Brozek and Bourgoin, 2013), where they are uniporous sensilla with terminal pore and flexible socket, and have been shown to serve mechanoreceptive or contact-chemoreceptive functions based on ultrastructural studies (Altner and Prillinger, 1980; Zacharuk, 1980; _ Foster et al. 1983; Brozek and Bourgoin, 2013). Currently, it is uncertain whether the sensilla basiconica have a terminal pore, although all are embedded in a flexible socket. From the above, although their function in Aphrophoridae feeding is not clear, possible hypotheses concerning their function include chemo- or mechanosensory, or both. Further studies are needed to examine variation in the structure of the labial tip of other genera to reveal the systematic importance of these characters and their usefulness in phylogenetic analysis. Multi-peg structures were found on the apical labial segment of some Gerromorpha in Heteroptera (Cobben, 1978; Cranston and _ Sprague, 1961; Brozek and Zettel, 2014). The function of these multi-peg structures is uncertain though speculated to be a sensory function (Cranston and Sprague, 1961) or a mechanical function, recording friction between mandibles during sucking (Cobben, 1978). In P. albinotata, multi-peg structures were found on each side of the labial opening arranged almost in a semicircle. The multi-peg structures located at the distal labial segment in Gerridae _ were identified by Brozek and Zettel (2014) but their function was not discussed. Based on their morphology and position, multi-peg structures are probably involved in the cleaning of stylet bristles and perception of stylet position. The feeding mechanism may be inferred from the mouthpart morphology of insects. Based on its structure, the labrum is probably not moveable in P. albinotata Uhler. When they feed, they stretch out the labial segments and anchor the labial tip on the plant surface. After locating a feeding site using sensilla, the labium bends and the stylet fascicle stretches out. The sharp end and serrate edges of the mandibular stylets pierce the plant cuticle and epidermis while saliva is secreted. The left and right stylets penetrate alternately into a xylem vessel and anchor the stylets in place in the plant tissue, followed by protraction of the maxillary stylets. The maxillary stylets pierce the tissue to the required depth aided by enzymes in the watery saliva. As the stylets progress through the plant tissue, the insect secretes more of the viscous saliva that solidifies to form a close-fitting sheath around the stylet bundle. Through a series of backward and forward movements of the
9
stylets, the sheath is lengthened until the desired feeding site in the xylem has been reached. Then the maxillary stylets transport the fluid food to the mouth by a relatively air-tight food canal (Backus, 1988; Crews et al., 1998). In summary, this study revealed information about the mouthparts of P. albinotata, including some details of the fine structure not previously observed in Hemiptera. This will contribute to a better understanding of the sensory system and feeding behavior of spittlebugs. Additional comparative study will be needed to determine the functional and phylogenetic significance of some of the structures observed in P. albinotata that have not been reported in other hemipterans.
Acknowledgments We thank Chris Dietrich (Illinois Natural History Survey, Champaign, IL, USA), Chandra Viraktamath (Department of Entomology, University of Agricultural Sciences, India) and John Richard Schrock (Emporia State University, Emporia, KS, USA) for comments on an earlier draft of this paper. Thanks are also due to two Reviewers who have greatly improved the manuscript with their suggestions. This project was supported by the National Natural Science Foundation of China (Nos. 30970385, 31093430, 31272343), “Program for New Century Excellent Talents in University (NCET10-0690)”, and the “Chinese Universities Scientific Fund (YQ2013010)”, and partly also by “the West Light Foundation of the Chinese Academy of Sciences (2012DF06)”.
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Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae) Tingting Wang, Liuxing Pan, Yalin Zhang, Wu Dai* Key Laboratory of Plant Protection Resources and Pest Management of the Ministry of Education, Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 August 2014 Accepted 5 December 2014 Available online xxx
Mouthparts associated with feeding behavior and feeding habits are important sensory and feeding structures in insects. To obtain a better understanding of feeding in Cercopoidea, the morphology of mouthparts of the spittlebug, Philagra albinotata Uhler was examined using scanning electron microscopy. The mouthparts of P. albinotata are of the typical piercingesucking type found in Hemiptera, comprising a cone-shaped labrum, a tube-like, three-segmented labium with a deep groove on the anterior side, and a stylet fascicle consisting of two mandibular and two maxillary stylets. The mandibles consist of a dorsal smooth region and a ventral serrate region near the apical half of the external convex region, and bear five nodules or teeth on the dorsal external convex region on the distal extremity; these are regarded as unique features that distinguish spittlebugs from other groups of Hemiptera. The externally smooth maxillary stylets, interlocked to form a larger food canal and a smaller salivary canal, are asymmetrical only in the internal position of longitudinal carinae and grooves. One dendritic canal is found in each maxilla and one in each mandible. Two types of sensilla trichodea, three types of sensilla basiconica and groups of multi-peg structures occur in different locations on the labium, specifically the labial tip with two lateral lobes divided into anterior sensory fields with ten small peg sensilla arranged in a 5 þ 4 þ 1 pattern and one big peg sensillum, and posterior sensory fields with four sensilla trichodea. Compared with those of previously studied Auchenorrhyncha, the mouthparts of P. albinotata may be distinguished by the shape of the mandibles, the multi-peg structures and a tooth between the salivary canal and the food canal on the extreme end of the stylets. The mouthpart morphology is illustrated using scanning electron micrographs, and the taxonomic and putative functional significance of the different structures is briefly discussed. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Hemiptera Philagra albinotata Mouthparts Sentilla Morphology
1. Introduction Cercopoidea, the second most species-rich superfamily in the Cicadomorpha, comprises approximately 3000 described species. The superfamily has been classified into five families: Cercopidae, Aphrophoridae, Clastopteridae, Machaerotidae and Epipygidae (Cryan and Svenson, 2010). Adults feed on the leaves and stems of a variety of plants by inserting their maxillary stylets into the xylem elements and suck the sap (Leopold et al., 2003). The saliva of some species, such as the sugarcane spittlebug, Mahanarva fimbriolata, contains toxic enzymes and feeding by these insects on plants may
* Corresponding author. Tel.: þ86 29 87092509; fax: þ86 29 87092190. E-mail address: [email protected] (W. Dai).
result in the blockage of conducting channels causing dessication of the leaves (Nunes and Camargo-Mathias, 2006). Mouthparts are important sensory and feeding structures in insects and their structure is closely associated with feeding behavior and feeding habits (Ma et al., 2013). Understanding the structure of mouthparts and accompanying sensilla is relevant, not only in host plant selection studies, but also in the study of feeding behavior and sensory physiology (Rani and Madhavendra, 2005). In addition, because mouthpart structures may be characteristic of all members of a genus, family or order of insects, knowledge of mouthpart morphology is useful for classification and identification (Gullan and Cranston, 2005). Previous work on the ultrastructural morphology of Hemiptera mouthparts, using light and scanning electron microscopy, has mainly focused on Heteroptera (Anderson et al., 2006; Boyd, 2003; Cobben, 1978), Aphidoidea (Forbes, 1969;
http://dx.doi.org/10.1016/j.asd.2014.12.001 1467-8039/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001
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Pollard, 1973; Razaq et al., 2000), Psyllidae (Garzo et al., 2012; Liang et al., 2013) and Aleyrodidae (Rosell et al., 1995; Walker and Gordh, 1989) of Sternorrhyncha, and Cicadellidae (Tavella and Arzone, 1993; Leopold et al., 2003; Wiesenborn, 2004; Zhao et al., 2010; Ammar and Hall, 2012) and Fulgoroidea (Brentassi and de Remes Lenicov, 2007; Dai et al., 2014; Liang, 2005) of Auchenorrhyncha. Apart from some brief notes on the stylets of Philaenus spumarius (L.) (Pollard, 1971) and Notozulia entreriana (Berg) (Paladini et al., 2008), and the interlocking pattern of maxillae and mandibles of n) (Cobben, 1978), fine structure of the Aphrophora alni (Falle mouthparts in spittlebugs has received little attention. Information on the distribution of the sensilla on the mouthparts, the relationship between mouthpart structure and function in feeding and the utility of this information for classification of spittlebugs is not available. In the present work, the mouthpart morphology of Philagra albinotata Uhler is described to provide comparative data elucidating variation within the family, contribute to knowledge of feeding behavior, and also to reveal characters that may be useful for future studies in taxonomy and phylogeny of Auchenorrhyncha, especially within Cercopoidea. 2. Material and methods 2.1. The insect Adult P. albinotata were collected at Huoditang in Ningshan, Shaanxi Province, China in July 2011, using a sweep net and preserved in 75% ethanol. Voucher specimens were deposited in the Entomological Museum of Northwest A&F University, Yangling, Shaanxi Province, China. 2.2. Sample preparation for SEM The heads of randomly selected five female and male specimens respectively were freed from the body. The mouthparts were excised and dissected using fine dissecting needles under 40 magnification (Nikon SMZ 1500, stereomicroscope, Japan). Specimens were then fixed with 2.5% glutaraldehyde for 12 h at 4 C, followed by washing in phosphate buffered saline (PBS, 0.1 M, pH7.2) for 15 min four times and cleaned in an ultrasonic cleaner for 15 s three times before dehydration in a graded series of 30%, 50%, 70%, 80%, 90% and 95% ethanol for 20 min each and 100% ethanol for 30 min twice. Specimens then underwent replacement in the mixture of 100% alcohol and 100% tertiary butyl alcohol mixed in sequential ratios (3:1, 1:1 and 1:3 by volume) for 15 min each time. Samples were then soaked in 100% tert-Butanol for 30 min before being placed into a freeze-drier with liquid CO2 for 3 h. Thereafter, the samples were mounted on aluminum stubs with double-sided copper sticky tape and sputtered with gold/ palladium (40/60) in a LADD SC-502 (Vermont, USA) high resolution sputter coater. The samples were subsequently examined with a Hitachi S-3400N scanning electron microscope operated at 15 kV (Zhao et al., 2010). All measurements are given as mean ± S.E. 3. Results 3.1. Gross morphology of mouthparts As in other Auchenorrhyncha, the mouthparts of Philagra albinotata arise from the posteroventral part of the head capsule (Fig. 1A) and are typical piercingesucking form (Fig. 1A and B) composed of the labrum (Lm) (Figs. 1B and 2B), a tube-like labium (Lb) (Fig. 1) and a stylet fascicle (Sf) consisting of two mandibular
(Md) and two maxillary stylets (Mx). The three-segmented labium has a longitudinal groove in the middle of the venter, called the labial groove (Lg) (Fig. 1B), which envelops the stylet fascicle comprising two inner maxillary stylets partially surrounded by two shorter and serrate-edged mandibular stylets, and proximally by the small cone-shaped labrum. The surface of the labium has different types of sensilla symmetrically arranged on both sides of the labial groove or positioned on the labial apex (Fig. 1B). 3.2. Labrum The cone-shaped labrum (Lm) is very short (411 ± 6 mm, n ¼ 5) (Fig. 2A and B), and is attached to the anterior margin of the anteclypeus and overlies the 1st and 2nd labial segments (Figs. 1B and 2A). The surface of the labrum is covered with a few scattered short triangular spines (Fig. 2C). Some clusters of pegs on the lateral edge and the terminus are more distinct and palmate (Fig. 2C). 3.3. Labium The labium (Lb) (also known as the rostrum or proboscis) is tubular in shape and composed of three segments (Fig. 1C). The anterior surface of the labium is bisected by a deep labial groove (Lg) (Fig. 1B), extending its entire length, enclosing two mandibular and two maxillary stylets. Sensilla are mainly distributed symmetrically on each side of the labial groove (Lg) and distally, but fewer occur on the dorsal and lateral surface (Fig. 1B and C). The tip of the labial segment is flattened with an opening from which the apices of the stylets are extended (Figs. 1B, C, and 5B). The total length of the labium is 2109 ± 20 mm (n ¼ 5). It is broad and of uniform width through most of its length with the distal segment widening near the tip. The first labial segment is the shortest (567.3 ± 9.6 mm, n ¼ 5) with most of it concealed by the overlapping anteclypeus (Fig. 1B). There are scarcely any sensilla trichodea I on this segment (Fig. 1C). The second labial segment is the longest (781 ± 17 mm, n ¼ 5) of the three segments (Figs. 1C and 2A), with a small round tumid area close to the first segment (Fig. 3A). There are two types of sensilla, sensilla trichodea I and sensilla basiconica I. Most sensilla trichodea I (s.t.I) are arranged symmetrically on each side of the labial groove (Fig. 2A and D) and on the lateral surface, and a few are arranged on posterior surface near the junction of the middle and third segments (Fig. 3A). Sensilla trichodea I, ranging from 75 mm to 140 mm in length, are slender and slightly curved with pointed tips. The surface has longitudinal grooves that spiral slightly around the shaft, and gradually converge on the obverse side, gradually disappearing near the distal part (Fig. 2D and E). Sensilla basiconica I (<30 mm in length) are short, stout basiconic pegs with a blunt tip, with a minute longitudinal groove in the shaft, arise from sunken pits (Fig. 2D), and are randomly distributed on the ventrum and dorsum of the labium. Numerous clusters of short denticles are scattered over the anterior surface of second segment (Figs. 2A and 3A). The third labial segment is 761 ± 5 mm (n ¼ 5) long, tubular, of uniform width from base to apical 1/4 then widens to the apex (Figs. 1C, 3B and D). There are some sensilla trichodea I and several sensilla basiconica I arranged on each side of the labial groove and randomly distributed on the dorsum of the labium (Fig. 3B, D, F and G). Two sensilla basiconica II are present on each side of the junction of the second and third segments (Fig. 3D and E). The labial tip forms two lateral lobes separated by the anterior stylet groove and posterior groove. Each lateral lobe has an anterior sensory field located laterad of the stylet groove and a posterior sensory field located behind the stylet groove (Fig. 4C). Each
Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001
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Fig. 1. Scanning electron micrographs showing the head including the mouthparts. (A) SEM micrograph of the head of adult Philagra albinotata showing the position of the mouthparts in anteroventral view. (B) Anterior view of labrum and labium showing sensilla symmetrically located on each side of the labial groove (Lg). (C) Dorsal view of mouthparts showing three-segmented labium (IeIII). Bars: A ¼ 1 mm; B and C ¼ 500 mm.
anterior sensory field consists of two kinds of short, stout basiconic pegs (Fig. 4C). The larger one is sensilla basiconica III (S.b.III) at the center of each side of the labial opening, with a bulbous base, and measuring 60 mm in length (Fig. 4B and C). This bristle is straight with a relatively blunt tip (Fig. 4B and C). Ten smaller sensilla basiconica II (S.b.II) (Fig. 4C and E) are blunt tipped, 25 mme28 mm in length, and located on either side of the labial groove, arising from a slightly raised flexible socket which tightly embraces the base of the shaft (Fig. 4C and E). The cuticular walls bear longitudinal grooves with a molting pore near the base. The sensilla basiconica II are divided into three groups, numbered 1, 5 and 4 from anterior to posterior, respectively. The posterior sensory field consists of two long tapering sensilla trichodea II (S.t.II) on each side of the posterior groove (Fig. 4A and D). The sensilla trichodea II are 75.5 ± 0.7 mm (n ¼ 8) in length and are symmetrically located on either side near the tip of the labium (Fig. 4A and D). They are straight, tapered setae with sharp apices and arise from a slightly raised flexible socket which tightly embraces the base of the shaft, and its surface bears longitudinal grooves with a cavity near the base (Fig. 4D). There are multi-peg structures (MS) on the ventral margin of stylet opening of the labial tip (Figs. 3B, C, 4A and C).
These structures include numerous tall (tp) and short (sp) pegs, grouped on the ventral edge of the opening of the labial tip (Fig. 3C). The short pegs are mostly located on the outer edge of the cluster, away from the labial tip (Fig. 3C). The pegs of these structures are directed towards the end of the terminal segment. 3.4. Stylet fascicle The needle-like stylet fascicle (Sf) comprising two mandibular (Md) and two maxillary stylets (Mx) (Fig. 5A) is housed in the labium and protrudes from the flattened labial tip (Fig. 5B). The mandibular (Md) stylets are crescent-shaped in cross section, located laterad of each maxillary stylets and are slightly shorter than the maxillary stylets (Fig. 5B). The inner surface of the mandibular stylet is smooth (Fig. 5E), to facilitate free movement of the maxillary stylets when probing the plant tissue. On the dorsal margin of the convex external surface of each stylet tip are 4e6 nodules (Fig. 5C and D), which are among the most noticeable features of the mandibular stylet. The ventral margin of each mandibular stylet is serrate, with a corresponding row of obliquely transverse ridges, ranging from 5.7 mm to 7.2 mm (Fig. 5C, D and F),
Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001
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Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001
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which are prominent at the tip of the stylet and gradually reduced in a step-wise fashion toward the base. The cross-section of the stylet fascicle shows that each mandibular stylet has a dendritic canal (Fig. 5G), a large duct that runs the length of the stylet and is located centrally in the thickest portion of each structure (Fig. 5G). In contrast to the mandibles, the maxillary stylets are not bilaterally symmetrical but are more complex (Fig. 5B). The external surface of the maxillary stylets is smooth (Fig. 5H). The two stylets are held together by internal longitudinal grooves and their interlocking mechanism. The two internal longitudinal grooves interlock with each other forming a food canal (Fc) which is used to suck plant juice, and a salivary canal (Sc) that delivers saliva to the plant (Fig. 5G and I). The two maxillary stylets in cross-section form approximately an ellipse due to the joining of the two crescentshaped sides. The hollow food canal is approximately circular in cross section and located in the center. The salivary canal (Sc) is also circular and located laterally on the inner side of one of the joints where the stylets interlock (Fig. 5G and I). The diameter of the food canal is 30.8 ± 0.5 mm (n ¼ 4), which is much larger than the salivary canal of 8.0 ± 0.2 mm (n ¼ 4) in diameter. On the extreme end of the stylets, the salivary canal has a controllable opening which is adjusted by a tooth and connects with the food canal through which it may deliver a small portion of secreted saliva to digest plant juices or sap (Fig. 5I). Within each maxillary stylet there is one approximately semicircular dendritic canal, smaller than the food canal, but larger than the salivary and dendritic canals in the mandibular stylet (Fig. 5G). These canals are distinct from the food and salivary canals. 4. Discussion We provide the first description of the fine structure of the mouthparts of the spittlebug. There are a number of morphological similarities in the mouthparts of this insect to those of Auchenorrhyncha species described previously (Anderson et al., 2006; Boyd, 2003; Forbes, 1969; Pollard, 1973; Garzo et al., 2012; Liang et al., 2013; Rosell et al., 1995; Zhao et al., 2010). The basic structure of the mouthparts consists of a cone-shaped labrum covered with some triangular spines, a cylindrical, three-segmented labium with abundant different types of sensilla mainly symmetrically distributed on each side of the labial groove, a needle-like stylet fascicle comprising two mandibular and two maxillary stylets, a crescentshaped mandibular stylet with several serrate ridges (Sr) on the surface of each stylet tip and one row of numerous tooth-like projections on the lateral edge of each stylet and two maxillary stylets held together forming a food canal (Fc) and a salivary canal (Sc). The basic similarities in the piercingesucking mouthparts of Auchenorrhyncha are likely due to the similarity in their feeding habits (Zhao et al., 2010). Previous study of the fine structure and function of the labrum has apparently been limited to two leafhopper species, Psammotettix striatus (L.) and Homalodisca vitripennis (as Homalodisca coagulata), and one planthopper species, Sogatella furcifera (Dai et al., 2014; Leopold et al., 2003; Zhao et al., 2010). Although there are some common features among hemipteran insects, the shape, size, and density and distribution of the triangular spines on the surface are clearly different in the species studied. The surface of the labrum in Sogatella furcifera (Hemiptera: Delphacidae) is covered with numerous small irregular papillae, and its lateral side
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has sparsely distributed sensilla trichodea (Dai et al., 2014). The labrum in Psammotettix striatus (L.) and Homalodisca vitripennis have small triangular cuticular processes and no sensilla on them, while the surface is smooth in Cacopsylla chinensis, and a sensillum basiconicum I is located on each side of the junction of the labrum and anteclypeus (Dai et al., 2014; Leopold et al., 2003; Liang et al., 2013; Zhao et al., 2010). In our research, the surface of the labrum in P. albinotata is covered with scattered short triangular cuticular processes. Based on the characteristics of the labrum, Aphrophoridae are most similar to Cicadellidae. The sharp end and the abundant protrusions of the mandibular stylets are structures specialized to pierce plant tissues while probing and are also present in other hemipteran insects (Forbes, 1969; Ullman and McLean, 1986; Zhao et al., 2010; Garzo et al., 2012). The limited variation observed among Auchenorrhyncha in the shape of the stylets may be due to the strong functional constraints on these structures and reflects the similar phytophagous feeding habits among species within the group. However, some details of the fine structure differ and these require additional comparative study to determine their functional and phylogenetic significance. In P. albinotata, on the medial surface of the tip of each mandibular stylet, there is a row of ridges (Sr) giving the stylet a serrate appearance. These structures presumably are specialized for perforating plant tissues using stylet movement and the extent of their development may be related to the depth of stylet penetration into the host plant (Garzo et al., 2012; Leopold et al., 2003; Liang et al., 2013; Rani and Madhavendra, 2005; Zhao et al., 2010). Such ridges are also found in other hemipteran insects, but differ in number and pattern among taxa. Each mandibular stylet has five teeth in Delphacidae (Dai et al., 2014) and ten teeth are present in Psyllidae (Garzo et al., 2012; Liang et al., 2013; Pollard, 1970; Ullman and McLean, 1986), whereas more than ten ridges may be found on the mandibles of Aphididae (Forbes, 1969) and nine to twenty teeth occur in different species of leafhoppers (Leopold et al., 2003; Zhao et al., 2010). The teeth are sharper in predacious hemipterans than in phytophagous hemipterans (Anderson et al., 2006; Boyd et al., 2002; Boyd, 2003; Garzo et al., 2012). In P. albinotata, except for five dorsal nodules, numerous ridges on the apical half of the stylet are restricted to the ventral part of the external convex region while the dorsal part is smooth. These structures have not been observed in other studied hemipteran taxa. Study of additional spittlebug species, as well as other hemipterans, is needed to determine whether the serrate mandibular stylet is a unique feature of Cercopoidea distinguishing them from other Hemiptera species. Study of other xylem-feeding Auchenorrhyncha such as cicadas is especially needed in order to determine whether this feature is associated with xylem feeding. Maxillary stylets were observed to be asymmetrical only in the internal position of longitudinal carinae and grooves, and not in their apical shape. As in previously studied leafhoppers and planthoppers, the grooves on the maxillary stylets of P. albinotata Uhler form a salivary canal (Sc) and a food canal (Fc). However, a tooth between the salivary canal and the food canal on the extreme end of the stylets is present in P. albinotata but has not been observed in other Auchenorrhyncha. Based on its morphology and position, this tooth may allow for the saliva to enter the food canal. The prominent dendritic canals within the mandibular and maxillary stylets demonstrate that the dual innervation of the fascicle is extensive and, based on previous reports, probably has a
Fig. 2. SEM of Philagra albinotata. (A) Anterior view of labrum and second labial segment. (B) The cone-shaped labrum (Lm). (C) Enlarged view of outlined box of (B) showing triangular spines (black arrow) and clusters of pegs (white arrow). (D) Anterior view of apical half of the second labial segment showing sensilla trichodea I (S.t.I) and sensilla basiconica I (S.b.I). (E) Enlarged view of sensillum trichodeum I (S.t.I) showing longitudinal grooves that spiral slightly around the shaft and gradually converge on the obverse side. Bars: A ¼ 80 mm; B ¼ 60 mm; C ¼ 8 mm; D ¼ 60 mm; E ¼ 25 mm.
Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001
Fig. 3. (A) Dorsal view of second labial segment showing a small round tumid area (black arrow), sensilla trichodea I (S.t.I) and sensilla basiconica I (S.b.I). (B) Dorsal view of the third labial segment showing sensilla trichodea I (S.t.I), sensilla basiconica I (S.b.I) and multi-peg structures (MS). (C) Enlarged view of multi-peg structures (MS), showing tall (tp) and short (sp) pegs. (D) Anterior view of the third labial segment showing sensilla trichodea I (S.t.I) and sensilla basiconica II (S.b.II). (E) Enlarged view of sensilla basiconica II (S.b.II). (F) Enlarged view of sensilla trichodea I (S.t.I) and sensilla basiconica I (S.b.I). (G) Enlarged view of outlined box of (D) showing sensilla trichodea I (S.t.I) and sensilla basiconica I (S.b.I). Bars: A ¼ 100 mm; B ¼ 150 mm; C ¼ 12 mm; D ¼ 160 mm; E ¼ 6 mm; F ¼ 15 mm; G ¼ 60 mm.
Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001
T. Wang et al. / Arthropod Structure & Development xxx (2014) 1e10
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Fig. 4. SEM of Philagra albinotata labium. (A) Dorsal view of the apex of third labial segment showing sensilla trichodea II (S.t.II). (B) Ventral view of the apex of third labial segment. (C) Labial tip with stylet bundle removed, showing sensilla basiconica II (S.b.II) and sensilla basiconica III (S.b.III) from ventral side above. (D) Enlarged view of sensilla trichodea II (S.t.II). (E) Enlarged view of sensilla basiconica II (S.b.II). Bars: A ¼ B ¼ 60 mm; C ¼ 50 mm; D ¼ 20 mm; E ¼ 18 mm.
Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001
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T. Wang et al. / Arthropod Structure & Development xxx (2014) 1e10
Fig. 5. SEM of Philagra albinotata stylets. (A) Whole stylet fascicle. (B) Enlarged view of apex of stylet fascicle showing the outer mandibular stylets (Md) are slightly shorter than the maxillary stylets (Mx). (C) Mandibular stylet (Md) showing serrate ridge (Sr) on the convex external surface. (D) Tip of mandibular stylet (Md), showing serrate ridge (Sr) on the convex external surface and nodule (white arrow). (E) Smooth hollow inner surface in mandibular stylet (Md). (F) Enlarged view of serrate ridge (white arrow). (G) Cross-section of stylet fascicle showing mandibular stylets (Md) and maxillary stylets (Mx), the interlock between the maxillary stylets, food canal (Fc), salivary canal (Sc), one dendritic canal in each maxillary stylet (white asterisk) and one dendritic canal in each mandibular stylet (white asterisk). (H) Maxillary stylets (Mx) with smooth external surfaces. (I) One of the maxillary stylets (Mx) showing inner surface, food canal (Fc) and salivary canal (Sc). Bars: A ¼ 300 mm; B ¼ 60um; C ¼ 15 mm; D ¼ 20 mm; E ¼ 30 mm; F ¼ 12 mm; G ¼ 15 mm; H ¼ 30 mm; I ¼ 15 mm.
Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001
T. Wang et al. / Arthropod Structure & Development xxx (2014) 1e10
proprioceptive function (Foster et al., 1983; Leopold et al., 2003; Zhao et al., 2010). The number of dendritic canals in the maxilla and mandible varies in different species. The studies of Backus and McLean (1982), Zhao et al. (2010), and Dai et al. (2014) clearly show that in Auchenorrhyncha 2e5 dendrites are present in each maxilla and 1e3 in each mandible. In P. albinotata, each mandible has one dendritic canal and each maxilla also has one dendritic canal. Three types of sensilla cover the tip of the labium of the spittlebug species: sensilla trichodea II, sensilla basiconica II and sensilla basiconica III. Although the functions of the sensilla on the labium have not been determined, it has been reported for several hemipterans that such rostral sensilla may respond to different stimuli produced by the host plant, and most of them are chemoreceptors and mechanoreceptors (Backus and McLean, 1982; Brozek and Bourgoin, 2013; Foster et al., 1983; Rani and Madhavendra, 2005; Zhao et al., 2010). The sensilla basiconica and the poreless sensilla trichodea come into direct contact with the host plant surface. The sensilla basiconica III in P. albinotata are extremely similar to those of Homalodisca vitripennis, which are regarded as mechanoreceptors (Leopold et al., 2003). Similar apical labial sensilla have been reported in other leafhoppers (Backus and McLean, 1982; Zhao et al., 2010). Ten sensilla basiconica II located in the dorsal margin of the tip of the labium are similar to those of _ Fulgoromorpha (Foster et al., 1983; Backus, 1985; Brozek and Bourgoin, 2013), where they are uniporous sensilla with terminal pore and flexible socket, and have been shown to serve mechanoreceptive or contact-chemoreceptive functions based on ultrastructural studies (Altner and Prillinger, 1980; Zacharuk, 1980; _ Foster et al. 1983; Brozek and Bourgoin, 2013). Currently, it is uncertain whether the sensilla basiconica have a terminal pore, although all are embedded in a flexible socket. From the above, although their function in Aphrophoridae feeding is not clear, possible hypotheses concerning their function include chemo- or mechanosensory, or both. Further studies are needed to examine variation in the structure of the labial tip of other genera to reveal the systematic importance of these characters and their usefulness in phylogenetic analysis. Multi-peg structures were found on the apical labial segment of some Gerromorpha in Heteroptera (Cobben, 1978; Cranston and _ Sprague, 1961; Brozek and Zettel, 2014). The function of these multi-peg structures is uncertain though speculated to be a sensory function (Cranston and Sprague, 1961) or a mechanical function, recording friction between mandibles during sucking (Cobben, 1978). In P. albinotata, multi-peg structures were found on each side of the labial opening arranged almost in a semicircle. The multi-peg structures located at the distal labial segment in Gerridae _ were identified by Brozek and Zettel (2014) but their function was not discussed. Based on their morphology and position, multi-peg structures are probably involved in the cleaning of stylet bristles and perception of stylet position. The feeding mechanism may be inferred from the mouthpart morphology of insects. Based on its structure, the labrum is probably not moveable in P. albinotata Uhler. When they feed, they stretch out the labial segments and anchor the labial tip on the plant surface. After locating a feeding site using sensilla, the labium bends and the stylet fascicle stretches out. The sharp end and serrate edges of the mandibular stylets pierce the plant cuticle and epidermis while saliva is secreted. The left and right stylets penetrate alternately into a xylem vessel and anchor the stylets in place in the plant tissue, followed by protraction of the maxillary stylets. The maxillary stylets pierce the tissue to the required depth aided by enzymes in the watery saliva. As the stylets progress through the plant tissue, the insect secretes more of the viscous saliva that solidifies to form a close-fitting sheath around the stylet bundle. Through a series of backward and forward movements of the
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stylets, the sheath is lengthened until the desired feeding site in the xylem has been reached. Then the maxillary stylets transport the fluid food to the mouth by a relatively air-tight food canal (Backus, 1988; Crews et al., 1998). In summary, this study revealed information about the mouthparts of P. albinotata, including some details of the fine structure not previously observed in Hemiptera. This will contribute to a better understanding of the sensory system and feeding behavior of spittlebugs. Additional comparative study will be needed to determine the functional and phylogenetic significance of some of the structures observed in P. albinotata that have not been reported in other hemipterans.
Acknowledgments We thank Chris Dietrich (Illinois Natural History Survey, Champaign, IL, USA), Chandra Viraktamath (Department of Entomology, University of Agricultural Sciences, India) and John Richard Schrock (Emporia State University, Emporia, KS, USA) for comments on an earlier draft of this paper. Thanks are also due to two Reviewers who have greatly improved the manuscript with their suggestions. This project was supported by the National Natural Science Foundation of China (Nos. 30970385, 31093430, 31272343), “Program for New Century Excellent Talents in University (NCET10-0690)”, and the “Chinese Universities Scientific Fund (YQ2013010)”, and partly also by “the West Light Foundation of the Chinese Academy of Sciences (2012DF06)”.
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Please cite this article in press as: Wang, T., et al., Morphology of the mouthparts of the spittlebug Philagra albinotata Uhler (Hemiptera: Cercopoidea: Aphrophoridae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/j.asd.2014.12.001