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Sensory armature and stylets of the mouthparts of Stephanitis nashi (Hemiptera: Cimicomorpha: Tingidae), their morphology and function
Journal Pre-proof Sensory armature and stylets of the mouthparts of Stephanitis nashi (Hemiptera: Cimicomorpha: Tingidae), their morphology and function Yan Wang, Jolanta Bro˙zek, Wu Dai
PII:
S0968-4328(19)30362-2
DOI:
https://doi.org/10.1016/j.micron.2020.102840
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JMIC 102840
To appear in:
Micron
Received Date:
14 November 2019
Revised Date:
20 January 2020
Accepted Date:
22 January 2020
Please cite this article as: Wang Y, Bro˙zek J, Dai W, Sensory armature and stylets of the mouthparts of Stephanitis nashi (Hemiptera: Cimicomorpha: Tingidae), their morphology and function, Micron (2020), doi: https://doi.org/10.1016/j.micron.2020.102840
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Sensory armature and stylets of the mouthparts of Stephanitis nashi (Hemiptera: Cimicomorpha: Tingidae), their morphology and function Yan Wanga, Jolanta Brożekb, Wu Daia, *
a
Key Laboratory of Plant Protection Resources and Pest Integrated Management of the Ministry of Education, College of Plant Protection, Northwest A&F University, Yangling, 712100, Shaanxi, China; [email protected](Y.W.); [email protected]
b
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(W.D.) Faculty of Natural Science, Institute of Biology, Biotechnology and Environmental
Protection, University of Silesia in Katowice, Bankowa 9, 40-007 Katowice, Poland;
Correspondence to: Wu Dai
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*
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[email protected]
Key Laboratory of Plant Protection Resources and Pest Integrated Management of the 712100, Shaanxi, China Tel.: +89-29-8708-2098
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Ministry of Education, College of Plant Protection, Northwest A&F University, Yangling,
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Email: [email protected]
Highlights
Ultramorphology of Stephanitis nashi mouthparts was examined for the first time.
Five kinds of sensilla were found at different locations on the
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labium.
Several unique features distinguish Tingidae from other groups of
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Hemiptera.
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Feeding mechanism was inferred from the mouthpart morphology
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of S. nashi
Abstract: Mouthparts are important appendages that are specialized for detection of food sources and feeding. The pear lace bug, Stephanitis nashi Esaki and Takeya, is a major pest
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of pear in China, sucking the sap and affecting plant growth. Fine structure of the mouthparts including distribution and abundance of receptor sensilla occurring of adult S.
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nashi was examined using scanning electron microscopy and structural details are described for the first time. The mouthparts of S. nashi are generally similar to those of other Hemiptera and consist of a pyramidal labrum, a tube-like segmented labium, and a stylet fascicle made up of two mandibular and two maxillary stylets. The four segments of the labium differ in length and have five classes of sensilla including 3 types of sensilla basiconica (I, II, III), 2 types of sensilla trichodea (I, II), 1 type of sensillum campaniformium, 1 type of flower-like sensillum and a sensillum placodeum. Sensilla trichodea II are distributed on each segment of the labium. Sensilla basiconica I occur on the
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base of the second and fourth segment. The labial tripartite apex composes of two sensory fields and a rostral lid. Each sensory field possesses 2 sensilla basiconica II, 9 sensilla basiconica III, 1 flower-like sensillum and 1 sensillum placodeum. The mandibular stylet tips have about 30 pairs of lateral minor teeth, which may help in penetrating leaves. Externally, the end of each maxillary stylet is smooth; internally it has five teeth. There is no obvious difference between males and females in the distribution, number and types of sensilla. The mouthparts morphology of S. nashi, is consistent in many respects to that of many other phytophagous hemipterans but appear to include some features unique to the Tingidae and others that closely resemble those of both phytophagous and predaceous
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Miridae.
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Keywords: Stephanitis nashi; Tingidae; mouthparts; sensillum; ultramorphology
1. Introduction Mouthparts are important appendages that are specialized for feeding and sense. Sensory structures on mouthparts are involved in feeding, locomotion and orientation (Lewis, 1970; Zacharuk, 1980). Some sensilla on the mouthparts and legs help insects to detect suitable foods (French et al., 2015). In insects, sensilla are traditionally classified into several groups based on their morphology (Slifer, 1970; Altner and Prillinger, 1980; Zacharuk, 1985). Functionally, they are usually divided
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into 4 groups, including chemo-, mechano-, hygro- and thermosensitive sensilla. Research of the ultrastructure of insect mouthparts may help elucidate the functions of sensilla and other structures and improve understanding of feeding mechanisms. Comparative study of such structures also may reveal important traits useful for inferring phylogenetic relationships (Faucheux et al., 2006; Brozek,
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2014).
Hemipteran bugs, recognizable by their piercing and sucking mouthparts, have evolved
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specialized morphological adaptations enabling them to exploit various food sources ranging from plant vascular fluids, to seed cell contents, to insect prey or vertebrate blood. Based on studies of
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comparative morphology (Bugnion and Popoff, 1908; Cobben, 1978) and ultrastructure of different labial and maxillary sensilla of the Heteroptera (Brożek, 2013; Brożek and Chłond; 2010; Brożek
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and Herczek, 2004; Brożek and Zettel, 2014), a remarkable range of modifications of the same basic sensory equipment is observed. The family Tingidae (lace bugs) is a member of the infraorder
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Cimicomorpha and, together with Miridae, form a monophyletic superfamily, Miroidea (Schuh and Štys, 1991; Schuh et al., 2009). All tingids are herbivorous, preferring ligneous plants (Schuh et al.,
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2009). Some tingids are important agro-forestry pests. The family presently contains more than 2500 described living species (Guilbert et al., 2014), in which only the antennal sensilla have been briefly described in several species (Lu et al., 2012; Xie et al., 2016). Although Tingidae is one of the largest families of plant-feeding Heteroptera, little information has been published on details of the mouthpart morphology in this family. In taxonomic papers, it is reported that the labium consists of four segments (Livingstone, 1969; Rodrigues, 1977; Péricart, 1983; Stehlik, 1997). Previous studies on tingid mouthparts concentrated on the stylet bundle
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(Cobben, 1978) of one species, Acalypta carinata (Panzer), in which the right maxillary stylet has several stout ventral teeth. The flattened and cross-striated apex of the mandibular stylets suggests that their principal function is probing deep feeding sites in the tissues of the host. Both pairs of stylets are reported to be equally involved in feeding by predominantly intracellular probing through the plant-tissue (Livingstone, 1969; Pollard, 1959), stomatal penetration (e.g. Stephanitis pyrioides (Scott))(Johnson, 1937; Ishihara and Kawai, 1981; Mathen et al., 1988) or penetration of the phloem (e.g. S. typica (Distant)) (Mathen et al., 1988). Lace bugs reduce photosynthesis in the leaves by destroying the palisade tissue, leading to chlorosis (Buntin et al., 1996). Details about the stylet
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interlocking mechanism of maxillae and mandibles of Dictyonata strichnocera Fieber, Acalypta gracilis (Fieber) and Dictyla echii (Schrank) were presented by Brożek and Herczek (2004). No other data on the mouthpart morphology (fine structure of maxillary and mandibular stylets) of tingid
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species are available. The sensilla of tingid mouthparts in particular have received very little attention (Cobben, 1978).
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The pear lace bug, Stephanitis nashi Esaki et Takeya (Hemiptera: Tingidae), an endemic species in East Asia, has been considered a minor pest of pear since it was described. But since the
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mid-1980s, populations of S. nashi have increased considerably in many provinces of China. The species is now regarded as a serious threat to orchards and gardens in East Asia (Zhang, 1985) and
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recent studies have mostly focused on its biological characteristics and integrated control (Xi et al., 1989; Xu et al., 2010; Wang et al., 2015). Mouthpart fine morphology of S. nashi may contribute to management of this pest by revealing more about its feeding behavior and mechanisms.
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The aim of this study was to describe the fine structure of the mouthparts of S. nashi. We focused
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on sensilla typology, distribution, quantity and possible functions and aimed to identify new character sets useful for future comparative morphological studies in Cimicomorpha. 2. Materials and Methods 2.1. Insect collecting Nine adult females and four adult males of S. nashi were obtained from the collection of the Hemiptera Morphology & Evolution Lab, College of Plant Protection, Northwest A&F University. 2.2. Samples for SEM
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All specimens of S. nashi were dissected and cleaned in an ultrasonic cleaner (KQ118, Kunshan, China). Dehydration in a series of successive ethanol solutions of 80%, 90%, 100% each for 15 min. The materials were air dried and then coated with a film of gold, and photographed with T-3400 SEM (Hitachi, Tokyo, Japan) operated at 15 kV or Nova Nano SEM-450 (FEI, Hillsboro, OR, USA) at 5– 10 kV. In the study was necessary to use the Nova Nano SEM-450 (FEI, Hillsboro, OR, USA) to show very small size and surface of sensilla (a pore system present or absent). 2.3. Image processing and morphometric measurement.
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Photographs and SEMs were observed and measured after being imported into Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA). 2.4. Terminology
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The terminology and classification of the labial sensilla is mainly based on the morphological criteria of Altner and Prillinger (1980), Zacharuk (1980), Brożek and Zettel (2014), and Brożek and
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Bourgoin (2013).
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3. Results 3. 1 Gross morphology of the mouthparts
The mouthparts of this species are similar to those of other heteropterans, consisting of a
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cone-shaped labrum, tubular segmented labium and stylet fascicle. The four labial segments differ in morphology and size (Table 1, Fig. 1A–1C). There are some sensilla symmetrically distributed on
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the labial surface including the apex. The labial tripartite apex is composed of two lateral lobes and a rostral lid. The lateral lobe has a sensory field bordered by a surrounding groove bearing 13 sensilla of
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different types (Fig. 6A–6C). The labrum is covered with numerous spike-like microtrichia (Fig. 2A and 2B). The mandibular stylet is covered with serrate ridges on the outer surface (Fig. 7C). Each maxillary stylet has a smooth external surface (Fig. 7F and 7G) and holds 2 internal grooves that together form a dorsal food canal (Fc) and ventral salivary canal (Sc), the right side being more developed (Fig. 7D and 7E). No obvious differences were noted in mouthpart structure and the length (p > 0.05) between females and males. The total length in females is 716.9 ± 32.6μm (n = 9), and for males is 702.5 ± 16.0μm (n = 4).
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3.1.1 Labrum The cone-shaped labrum, reaches half the length of the first labial segment (Fig. 1A and 2A). The basal part of labrum is wide but distal is elongated. Labrum morphologically divides into basilabrum (bl) strong sclerotized and distilabrum (dl) less sclerotized (Fig. 2A). Surface of the distilabrum is slightly an undulate. On ventral side of the distilabrum there is groove (gr), it keeps a proximal part of stylet fascicle (Sf) (Fig. 2B). Spike-like microtrichia (sm) are irregularly distributed throughout both surface labrum (dorsal and ventral) (Fig. 2A–2D). Four sensilla trichodea I (Fig. 2A and 2E) are also
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present. 3.1.2 Labium
The four labial segments are different in shape and size (Fig. 1A–1C). The first segment is stout, the base is broad and the end is narrowed. There is a bandlike dorsal plate (bdp) at the top that covers
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the joint between the two segments dorsally (Fig. 1B, 3A and 3B). Ventral membrane (vm) is well
distributed on the dorsal surface (Fig. 3A).
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developed between the first and the second segment (Fig. 1C and 3B). Sensilla trichodea II are
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The second segment is the longest, and the ends are relatively wide with a slight overlap in the middle (Fig. 4A and 4C). This segment contains three types of sensilla (sensilla basiconica I, sensilla trichodea II and sensilla campaniformia). There are three pairs of sensilla basiconica I at the base of
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the first sections (Fig. 3A and 4D). The dorsal, lateral and ventral surfaces bear some sensilla trichodea II (Fig. 4A–4C), and three pairs of sensilla campaniformia are irregularly arranged along
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the distal half of the second segment (Fig. 4A, 4F and 4G). The third labial segment is the shortest segment and is relatively broad (Fig. 4A–4C). In the
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proximal area of this segment on the anterior (dorsal) side there is small lobe (lo) arising on the left or right side and partly extended over the opposite side (Fig. 4A). The middle ventral part is slightly concave and two bands of undulated membrane (m1, m2) are visible (Fig. 4C). The distal end is convex ventrally (Fig. 4B and4C) and has few sensilla trichodea II on the dorsal, lateral and ventral surfaces (Fig. 4A–4C). The fourth, or distal, labial segment is tapered (Fig. 5A–5C). Several sensilla trichodea II are present laterally and ventrally. There is one pair of sensilla basiconica I at the base of the fourth
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segment. The labial tip is tripartite, distinctly divided into a rostral lid (rl) and two lateral lobes (Fig. 6A–6C). The rostral lid is formed by a small comb-like structure (Fig. 6A-6C). Two sensilla basiconica II (no 4, 9)(Fig. 6E), nine sensilla basiconica III (no 1-3, 5-8, 10-11), one flower-like sensillum (Fig. 6D) and one sensillum placodeum (Spl)(Fig. 6F) are found on the each of the sensory fields. The flower-like sensillum is present at the center of each lobe and an undulating, flower-like surface. A single sensillum placodeum is present at the side of each lobe.
3.1.3 Stylet fascicle
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The stylet fascicle is slender and composed of two separated mandibular stylets (Md) and two interlocked maxillary stylets (Mx) (Fig. 7A and 7B). The mandibular stylets (Md) are adpressed laterally to the maxillary stylets (Mx). The mandibular stylets have an internal concavity allowing them to precisely cover the maxillary stylets. The mandibular stylet tips have about 30 pairs of lateral
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small teeth (Sr), which may help in penetrating leaf tissue. Each maxillary stylet has 2 grooves that
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together form a dorsal food canal (Fc) and ventral salivary canal (Sc), the right side being more developed (Fig. 7D and 7E). The ends of the external surfaces of the maxillary stylets are smooth but
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on the inner ridges there are five small, slightly recurved teeth (Fig. 7D). The end of the right maxillary stylet is lancet-shaped (Fig. 7D–F) in contrast to the left stylet, which is blunt and apically slightly divided (Fig. 7E, G).
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The cross-sections of this lace bug species (Fig. 8) show that the two mandibular stylets are mirror symmetric and the two maxillary stylets are asymmetric. The mandibular and maxillary stylets
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are connected to each other through a T-shaped process. The two maxillary stylets have dorsal, middle and ventral inner locking systems (Fig. 8). The dorsal lock has two hooked process and two straight
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process. The middle lock has one T-shaped process and two hooked processes. The ventral lock has three hooked process and one straight process (Fig. 8). 3.2. Type and distribution of the mouthpart sensilla Under scanning electron microscopy five types of sensilla were observed on the mouthparts of adult males and females: sensilla basiconica (including three subtypes: Sb I, Sb II and Sb III), s. trichodea (including two subtypes: St I and St II), flower-like sensillum, s. campaniformia (Sca) and s.
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placodea (Spl), respectively. No consistent difference was found in abundance and distribution of sensilla between the male and female adults. 3.2.1 Sensilla trichodea Sensilla trichodea I (St I) are very long and slightly curved. The base of the sensilla have a flexible socket, the surface with a slight vertical groove and the tip is narrow and rounded (Fig. 2A and 2E). The length of St I is 58.13–90.79 μm, and the basal diameter is 2.23–2.57 μm. This type of sensillum was found on the labrum (distilabrum).
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Sensilla trichodea II (St II) are short and thick. The base of the sensilla has flexible socket and a slight vertical grooved surface (Fig. 3A, 3C, 3D, 4F, 5A–C). The length of St II is 11.42-19.47 μm, and the basal diameter is 1.43-2.02 μm. This type of sensillum occurs on the labium.
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3.2.2 Sensilla basiconica
Sensilla basiconica I (Sb I) are short, small and straight. The surfaces of sensilla are smooth and
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have a rounded tip (Fig. 3A, 4D and 4E). Sb I is 5.19–7.75 μm, and the basal diameter is 1.85–2.02
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μm. They are distributed between the first and second labial segments. Two sensilla basiconica II (Sb II) are present at the center of the apical sensory field (no 4, 9), and with a terminal pore and sit in a tightly fitting socket (Fig. 6E). Sb II is 2.58–5.36 μm, and the
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basal diameter is 1.18–1.42 μm.
Nine sensilla basiconica III (Sb III) are present at the center of the distal sensory field. Sb III is
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5.12–8.15 μm, and the basal diameter is 0.84-1.24. They are short, small, straight and have a smooth
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surface, a rounded tip and sit within a tightly fitting socket (Fig. 6C, no 1–3, 5–7, 10–11). 3.2.3 Sensilla campaniformia These are flat and oval shaped discs. Three pairs of sensilla campaniformia are distributed on the
second labial segment (Fig. 4F and 4G). The basal diameter is 4.63–6.13 μm. 3.2.4 Flower-like sensilla The flower-like sensillum is located in the middle of the lateral lobes of the labial tip (Fig. 6B). This sensillum is recognized as “flower-like sensillum” that consists of folds (fo) with very small
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pegs (pe) (Fig. 6D). Both structures are aporous, but probably the pegs have sensory function. The diameter of the undulated surface is 3.6–4.64μm. 3.2.5 Sensilla placodea These are oval shaped, elevated above the surface, with multiporous walls. This type of sensillum (Fig. 6F) occurs on each lateral lobe of the labial tip. The basal diameter is 3.16–3.44 μm. 4. Discussion Morphological studies of mouthpart fine structure reveal important information related to
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feeding behavior as well as evolutionary relationships. Scanning electron microscopy was used in this study to document previously understudied details of the mouthparts of Tingidae (Cimicomorpha) based on the economically important species S. nashi. Comparison of mouthpart structures of this
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species to that of other Heteroptera may help to explain modifications of these structures and their function.
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Labrum in most studied is very briefly discussed. According to Štys (1969) in Cimicomorpha types of the labrum have been recognized. One short, wide, extended labrum was found in few
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families. Other type of labrum as spiniform elongated and divided in basilabrum and distilabrum (often less sclerotized) was indicated e.g. in some Reduviidae. Latter type is similar to the tingid
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labrum of Stephanitis nashi.
The labium of tingids is four-segmented (Stehlik, 1997) as in most other heteropteran bugs (Schuh and Slater, 1995). Moreover, three specific labial characters were found in S. nashi: a dorsal
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bandlike plate and a ventral membrane are well developed between the first and the second segment,
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which affects the bend of labium during feeding (Fig. 3B). A similar bending mechanism is present in Pyrrhocoris sibiricus (Pyrrhocoridae) (Wang and Dai, 2017). The third segment in the ventral part is slightly sclerotized and mostly formed by a flexible/undulated membrane which influences flexibility during feeding. The long, stiff second segment holds the stylets and probably controls bending of the short third and fourth segments. The dorsal lobe of the third segment holds the stylets within the labial groove during feeding (Fig. 1A). This lobe is characteristic of Stephanitis nashi and has not been reported in other heteropterans.
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Labial
sensilla
and
their
functions
as
chemoreceptors,
mechanoreceptors
and
thermo-hygroreceptors were previously described in various hemipteran taxa (Brożek and Bourgoin, 2013; Rani and Madhavendra, 1995, 2005; Foster et al., 1983; Backus and McLean, 1982; Zhao et al., 2010). In S. nashi, two symmetrical fields on the labial tips are equipped with sensilla of three morphological types: two sensilla basiconica II (SbII), nine sensilla basiconica III (Sb III) and one multiporous sensillum placodeum (Spl); another flower-like sensillum is also present on each field. Variation in shape between two types of sensilla (Sb II and Sb III) in S. nashi may reflect same chemosensory functions. The single pore characterizing these sensilla is either terminal or
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sub-terminal suggesting a gustatory (Sb II) or contact-chemosensory function (Sb III gustative and touch). The latter are placed around a center of the labial tip and they are slightly longer than Sb II. Gustatory sensilla are mostly bimodal, comprising chemo- and mechanosensitive receptor cells
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(Zacharuk, 1980; Altner and Prillinger, 1980; Steinbrecht, 1992; Chapman, 1998; Shields, 2010).
Three pairs of sensilla campaniformia in S. nashi are distributed at the distal part of the dorsal
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surface of the second labial segment, they may function as proprioceptors responding to the stresses connected with the movement of the labium. Similar mechanosensilla were identified as
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proprioceptors in Peiratinae (Reduviidae) (Catalá, 1994), in the seed-bug, Pyrrhocoris sibiricus Kuschakevich (Wang and Dai, 2017) in phytophagous Pentatominae and in predatory Asopinae
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(Pentatomidae) (Parveen et al., 2015). It is common for proprioceptive sensilla campaniformia (Sca) to occur on mouthparts, antennae, bases of wings, halteres, legs, and eyes (Schneider, 1964; Bromley and Dunn, 1980; Wang et al., 2013; Zhang et al., 2016).
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The gustatory sensilla are generally abundant reflecting their important role in host location and
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evaluation. These sensilla are usually perpendicular to the labial tip and this position allows them easily to contact the substrate. Distribution and number of gustatory sensilla (9+2) in S. nashi resemble those of phytophagous mirids (e.g., 11 peg/basiconic sensilla in Lygus rugulipennis and L. lineolaris) (Avé et al., 1978; Hatfield and Frazier, 1980; Romani et al., 2005). S. nashi shares a similar pattern of gustatory sensilla with other plant bugs including the pentatomid Nezara viridula L. (Rani and Madhavendra, 1995) and the chinch bug Blissu sleucopterus leucopterus (Say) (Baker et al., 2008). However, the pattern is not stable and in some other plant bugs Dysdercus intermedius, D. fulvoniger and D. koenigii there are only 10 gustatory sensilla (Gaffal, 1981) while 9 such sensilla
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have been identified in the alydid Riptortus pedestris F. and 8 in the lygaeid Elasmolomus sordidus (F.) (Rani and Madhavendra, 2005). There is a significant difference in shape of the gustatory sensilla between S. nashi and predatory triatomine and peiratine assassin bugs (Reduviidae) (Brożek and Chłond, 2010; Catalá, 1996; Rosa et al., 1999). The latter have short uniporous peg sensilla corresponding to the sensilla basiconica of S. nashi, but reduviids also have dome-elongated uniporous sensilla (UDES) placed more subapically in relation to the peg sensilla. Similarly to some other cimicomorphans (Brożek and Chłond, 2010), the labial olfactory system in the studied species includes one multiparous sensillum. We hypothesize that this type of sensillum
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functions as an olfactory chemoreceptor, which may sense plant volatiles prior to feeding. It seems that in S. nashi (Tingidae) a typical sensillum placodeum may correspond to the sensillum basiconicum with a perforated wall in other phytophagous species, e.g., Dysdercus spp. and
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Pyrrhocoris sibiricus (Brożek and Zettel, 2014; Brożek and Bourgoin, 2013; Wang and Dai, 2017; Gaffal, 1981; Peregrine, 1972).
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An aporous flower-like sensillum is distributed at the center of each lobe. Aporous sensilla (e.g. sensilla basiconica and pegs) are associated with mechanoreception such as touch, thermo-, and
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hygroreception (McIver 1985). Aporous flower-like sensilla may be thermo-hygroreceptive in S. nashi. The fine structure of thermo-hygroreceptive aporous sensilla is conservative across insect
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orders (Altner et al., 1983; McIver, 1973; McIver and Siemicki, 1976; Steinbrecht and Müller, 1961; Yokohari, 1981). In S. nashi, aporous flower-like sensilla are located in the center of each lateral lobe and we provide the first detailed description.
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In S. nashi, the mandibular tips have at least 30 shallow serrations which extend somewhat
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transversely as also reported in A. carinata (Tingidae) (Cobben, 1978). The serrations are fewer in number (11) but deeper in D. nebulosus and other species of mirids (Boyd et al., 2002). Boyd et al. (2002) pointed out that the mandibular stylets of D. nebulosus are typical for Miridae regardless of whether they are phytophagous or predatory. The serrations on the mandibular stylet tip may be used to fix the stylets in host tissues (Cobben, 1978; Cohen, 2000; Wheeler, 2001). Different patterns of weak mandible serration (blunt and wide teeth) are present in other phytophagous species of Coreidae and Lygaeidae (Cobben, 1978; Gaffal, 1981; Cohen 1990; Depieri and Panizzi, 2010). These bugs produce a salivary flange that may act as a fulcrum for probe movement (Cohen, 1998, 2000).
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In this species the maxillary stylets (Mx) are smooth on the outer side but on the inner side are equipped with five small teeth directed away from the head (7D). The end of the right maxilla is lancet-shaped in contrast to a slightly divided end of the left maxilla. Additionally, the mandibular stylet tips are serrated with 30 pairs of wide and small teeth (Sr). Similar structures occur in another tingid species, Acalypta carinata, in which there are also five rudimentary teeth on the inner side of the maxillary stylet and several shallow indentations on the mandibular ends (Cobben, 1978). Maxillary stylets similar to S. nashi (Tingidae) were also observed in predatory species of mirids. Deraeocoris nebulosus and D. olivaceous have six teeth on the inner surface and D. nigritulus has
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seven teeth pointed distad (Cobben, 1978; Boyd et al., 2002; Boyd, 2003). The teeth in Deraeocoris spp. are slightly longer than those in S. nashi but the significance of such slight differences remain to be revealed by comparative study of additional species. The structure of the maxillary stylets of
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Miridae has been suggested to represent an intermediate condition between phytophagous and predatory types (Wheeler, 2001). Some observations suggest that the right maxillary stylets of
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predatory Heteroptera/Cimicomorpha (e.g., Nabidae, Anthocoridae, Reduviidae) are more deeply serrated and have more numerous barbs than those in phytophagous species in which the apices of the
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maxillae are only slightly serrated on the inner side (Tingidae, Lygaeidae, Pentatomidae, Pyrrhocoridae) (Khan, 1972; Cobben, 1978; Cohen, 1990; Wang and Dai, 2017). The orientation of
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the teeth on the maxillary stylets is also different. The maxillae of zoophagous heteropterans are armed with backward-curved teeth which better anchor the stylets in the tissues of struggling prey
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(Cobben, 1978; Cohen, 1996). 5. Conclusions
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Our detailed study of mouthpart fine structure in S. nashi represents the first comprehensive description of sensory structures of lace bug mouthparts. S. nashi (Tingidae) mouthparts show modifications similar to those found in phytophagous and zoophagous mirids. Morphological evidence indicates that many sensilla at the labial tip have gustatory and olfactory functions, consistent with their role in host plant selection.
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Acknowledgments: We are grateful to Chris Dietrich who reviewed the manuscript and offered several valuable suggestions for improvement. We also thank the Life Science Research Core Services of Northwest A&F University for providing scanning electron microscope. This project was supported by the National Natural Science Foundation of China (Nos. 31772514, 31572306, 31272343).
Conflicts of Interest: The authors declare no conflict of interest.
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Altner, H., Schaller-Selzer, L., Stetter, H., Wohlrab, I., 1983. Poreless sensilla with inflexible sockets - a comparative study of a fundamental type of insect sensilla probably comprising thermo- and hygroreceptors. Cell Tissue Res. 234, 279– 307. https://doi.org/10.1007/BF00213769.
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Avé, D., Frazier, J.L., Hatfield, L.D., 1978. Contact chemoreception in the tarnished plant bug Lygus lineolaris. Entomol. Exp. App. 24(3), 217–227.
Macrosteles
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Stål
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Figure legends
Figure 1. Scanning electron micrographs of the head of Stephanitis nashi. A. Dorsal view; B. Lateral view; C. Ventral view showing four-segmented labium (I–IV); Sf, stylet fascicle; Lm, labrum; Lb,
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labium; bdp, bandlike dorsal plate; vm,ventral membrane.
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Figure 2. SEM of labrum of Stephanitis nashi. A. Dorsal view of labrum (bl-basilabrum),
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dl-distilabrum), sensilla trichodea I (St I) on distilabrum; B. Ventral view of labrum (distilabrum); C. Enlarged view of tip of labrum; D. Enlarged view of box in (B); E.
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Enlarged view of sensillum trichodeum I (St I); sm, spike-like microtrichia; gr, groove; Sf,
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stylet fascicle.
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Figure 3. SEM of first labial segment of Stephanitis nashi. A. Lateral view of the first labial
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segment; B. Ventral view; C. Enlarged view of first labial segment; D. Enlarged view of
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sensillum trichodeum II (St II); bdp, bandlike dorsal plate; vm,ventral membrane.
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Figure 4. SEM of the second and third labial segment.A. Dorsal view; B. Lateral view; C. Ventral view; D. Enlarged view of box in (A), showing the base of the second segment; E. Enlarged view of sensillum basiconicum I (Sb I). F. Enlarged view of box in (A), showing the surface of the second segment; G. Enlarged view of sensillum campaniformium (Sca);
lo, lobe; m, membrane.
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Figure 5. SEM of the fourth labial segment of Stephanitis nashi. A. Dorsal view; B. Lateral
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view; C. Ventral view.
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Figure 6. SEM the tip of labium of Stephanitis nashi. A. Ventrolateral view; B. Apical view showing flower-like sensillum (black circles); C. Lateral view; D. Enlarged view of
flower-like sensillum; E. Enlarged view of sensillum basiconicum II-4 (SbII-4); F. Sensillum placodeum (Spl) (dome-shaped multiporous sensillum). rl, rostral lid; p, pore; fo, fold; pe, peg.
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Figure 7. SEM images of stylets of Stephanitis nashi. A. Enlarged ventral view of stylet fascicle apex; B. Enlarged ventral view of stylet fascicle showing mandibular stylets (Md) and maxillary stylets (Mx); C. Tip of mandibular stylets (Md) showing serrate ridge (Sr); D. Tip of inner surface of right maxillary stylet (RMx); E. Tip of the inner surface of left maxillary stylet (LMx); F. Enlarged view of tip of right maxillary stylet (RMx); G. Enlarged view of tip of left maxillary stylet (LMx); Fc, food canal; Sc, salivary canal.
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Figure 8. Diagram of cross-section of stylet fascicle of Stephanitis nashi. A, Hooked; A’,
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Straight; B, Straight; B’, Hooked; C, T-shaped; C’, Hooked; D, Hooked; D’, Hooked; E, Straight; E’, Hooked; CN, nerve canal; Fc, food canal; LMd, left mandibular stylet; LMx,
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left maxillary stylet; LPr, left process of the maxilla; RMd, right mandibular stylet; RMx,
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right maxillary stylet; RPr, right process of the maxilla; Sc, salivary canal.
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Table 1. Measurements of labrum and labium (mean ± SD) obtained from scanning electron microscopy. N = sample size. Lm, labrum; Lb, labium; Lb-sg1, first segment of labium; Lb-sg2, second segment of labium; Lb-sg3, third segment of labium; Lb-sg4, fourth segment of labium. Gender
Position
Length (μm)
Width (μm)
Male
Lb
702.5 ± 16.0
Female
Lm
193.2 ± 17.3
Lb
716.9 ± 32.6
Lb-sg1
213.8 ± 17.1
80.7 ± 4.0
9
Lb-sg2
269.0 ± 15.1
46.5 ± 2.7
9
Lb-sg3
114.3 ± 5.8
47.8 ± 6.2
9
Lb-sg4
119.8 ± 4.9
42.3 ± 4.1
9
4 74.4 ± 3.4
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PII:
S0968-4328(19)30362-2
DOI:
https://doi.org/10.1016/j.micron.2020.102840
Reference:
JMIC 102840
To appear in:
Micron
Received Date:
14 November 2019
Revised Date:
20 January 2020
Accepted Date:
22 January 2020
Please cite this article as: Wang Y, Bro˙zek J, Dai W, Sensory armature and stylets of the mouthparts of Stephanitis nashi (Hemiptera: Cimicomorpha: Tingidae), their morphology and function, Micron (2020), doi: https://doi.org/10.1016/j.micron.2020.102840
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Sensory armature and stylets of the mouthparts of Stephanitis nashi (Hemiptera: Cimicomorpha: Tingidae), their morphology and function Yan Wanga, Jolanta Brożekb, Wu Daia, *
a
Key Laboratory of Plant Protection Resources and Pest Integrated Management of the Ministry of Education, College of Plant Protection, Northwest A&F University, Yangling, 712100, Shaanxi, China; [email protected](Y.W.); [email protected]
b
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(W.D.) Faculty of Natural Science, Institute of Biology, Biotechnology and Environmental
Protection, University of Silesia in Katowice, Bankowa 9, 40-007 Katowice, Poland;
Correspondence to: Wu Dai
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*
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[email protected]
Key Laboratory of Plant Protection Resources and Pest Integrated Management of the 712100, Shaanxi, China Tel.: +89-29-8708-2098
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Ministry of Education, College of Plant Protection, Northwest A&F University, Yangling,
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Email: [email protected]
Highlights
Ultramorphology of Stephanitis nashi mouthparts was examined for the first time.
Five kinds of sensilla were found at different locations on the
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labium.
Several unique features distinguish Tingidae from other groups of
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Hemiptera.
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Feeding mechanism was inferred from the mouthpart morphology
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of S. nashi
Abstract: Mouthparts are important appendages that are specialized for detection of food sources and feeding. The pear lace bug, Stephanitis nashi Esaki and Takeya, is a major pest
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of pear in China, sucking the sap and affecting plant growth. Fine structure of the mouthparts including distribution and abundance of receptor sensilla occurring of adult S.
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nashi was examined using scanning electron microscopy and structural details are described for the first time. The mouthparts of S. nashi are generally similar to those of other Hemiptera and consist of a pyramidal labrum, a tube-like segmented labium, and a stylet fascicle made up of two mandibular and two maxillary stylets. The four segments of the labium differ in length and have five classes of sensilla including 3 types of sensilla basiconica (I, II, III), 2 types of sensilla trichodea (I, II), 1 type of sensillum campaniformium, 1 type of flower-like sensillum and a sensillum placodeum. Sensilla trichodea II are distributed on each segment of the labium. Sensilla basiconica I occur on the
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base of the second and fourth segment. The labial tripartite apex composes of two sensory fields and a rostral lid. Each sensory field possesses 2 sensilla basiconica II, 9 sensilla basiconica III, 1 flower-like sensillum and 1 sensillum placodeum. The mandibular stylet tips have about 30 pairs of lateral minor teeth, which may help in penetrating leaves. Externally, the end of each maxillary stylet is smooth; internally it has five teeth. There is no obvious difference between males and females in the distribution, number and types of sensilla. The mouthparts morphology of S. nashi, is consistent in many respects to that of many other phytophagous hemipterans but appear to include some features unique to the Tingidae and others that closely resemble those of both phytophagous and predaceous
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Miridae.
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Keywords: Stephanitis nashi; Tingidae; mouthparts; sensillum; ultramorphology
1. Introduction Mouthparts are important appendages that are specialized for feeding and sense. Sensory structures on mouthparts are involved in feeding, locomotion and orientation (Lewis, 1970; Zacharuk, 1980). Some sensilla on the mouthparts and legs help insects to detect suitable foods (French et al., 2015). In insects, sensilla are traditionally classified into several groups based on their morphology (Slifer, 1970; Altner and Prillinger, 1980; Zacharuk, 1985). Functionally, they are usually divided
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into 4 groups, including chemo-, mechano-, hygro- and thermosensitive sensilla. Research of the ultrastructure of insect mouthparts may help elucidate the functions of sensilla and other structures and improve understanding of feeding mechanisms. Comparative study of such structures also may reveal important traits useful for inferring phylogenetic relationships (Faucheux et al., 2006; Brozek,
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2014).
Hemipteran bugs, recognizable by their piercing and sucking mouthparts, have evolved
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specialized morphological adaptations enabling them to exploit various food sources ranging from plant vascular fluids, to seed cell contents, to insect prey or vertebrate blood. Based on studies of
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comparative morphology (Bugnion and Popoff, 1908; Cobben, 1978) and ultrastructure of different labial and maxillary sensilla of the Heteroptera (Brożek, 2013; Brożek and Chłond; 2010; Brożek
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and Herczek, 2004; Brożek and Zettel, 2014), a remarkable range of modifications of the same basic sensory equipment is observed. The family Tingidae (lace bugs) is a member of the infraorder
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Cimicomorpha and, together with Miridae, form a monophyletic superfamily, Miroidea (Schuh and Štys, 1991; Schuh et al., 2009). All tingids are herbivorous, preferring ligneous plants (Schuh et al.,
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2009). Some tingids are important agro-forestry pests. The family presently contains more than 2500 described living species (Guilbert et al., 2014), in which only the antennal sensilla have been briefly described in several species (Lu et al., 2012; Xie et al., 2016). Although Tingidae is one of the largest families of plant-feeding Heteroptera, little information has been published on details of the mouthpart morphology in this family. In taxonomic papers, it is reported that the labium consists of four segments (Livingstone, 1969; Rodrigues, 1977; Péricart, 1983; Stehlik, 1997). Previous studies on tingid mouthparts concentrated on the stylet bundle
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(Cobben, 1978) of one species, Acalypta carinata (Panzer), in which the right maxillary stylet has several stout ventral teeth. The flattened and cross-striated apex of the mandibular stylets suggests that their principal function is probing deep feeding sites in the tissues of the host. Both pairs of stylets are reported to be equally involved in feeding by predominantly intracellular probing through the plant-tissue (Livingstone, 1969; Pollard, 1959), stomatal penetration (e.g. Stephanitis pyrioides (Scott))(Johnson, 1937; Ishihara and Kawai, 1981; Mathen et al., 1988) or penetration of the phloem (e.g. S. typica (Distant)) (Mathen et al., 1988). Lace bugs reduce photosynthesis in the leaves by destroying the palisade tissue, leading to chlorosis (Buntin et al., 1996). Details about the stylet
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interlocking mechanism of maxillae and mandibles of Dictyonata strichnocera Fieber, Acalypta gracilis (Fieber) and Dictyla echii (Schrank) were presented by Brożek and Herczek (2004). No other data on the mouthpart morphology (fine structure of maxillary and mandibular stylets) of tingid
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species are available. The sensilla of tingid mouthparts in particular have received very little attention (Cobben, 1978).
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The pear lace bug, Stephanitis nashi Esaki et Takeya (Hemiptera: Tingidae), an endemic species in East Asia, has been considered a minor pest of pear since it was described. But since the
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mid-1980s, populations of S. nashi have increased considerably in many provinces of China. The species is now regarded as a serious threat to orchards and gardens in East Asia (Zhang, 1985) and
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recent studies have mostly focused on its biological characteristics and integrated control (Xi et al., 1989; Xu et al., 2010; Wang et al., 2015). Mouthpart fine morphology of S. nashi may contribute to management of this pest by revealing more about its feeding behavior and mechanisms.
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The aim of this study was to describe the fine structure of the mouthparts of S. nashi. We focused
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on sensilla typology, distribution, quantity and possible functions and aimed to identify new character sets useful for future comparative morphological studies in Cimicomorpha. 2. Materials and Methods 2.1. Insect collecting Nine adult females and four adult males of S. nashi were obtained from the collection of the Hemiptera Morphology & Evolution Lab, College of Plant Protection, Northwest A&F University. 2.2. Samples for SEM
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All specimens of S. nashi were dissected and cleaned in an ultrasonic cleaner (KQ118, Kunshan, China). Dehydration in a series of successive ethanol solutions of 80%, 90%, 100% each for 15 min. The materials were air dried and then coated with a film of gold, and photographed with T-3400 SEM (Hitachi, Tokyo, Japan) operated at 15 kV or Nova Nano SEM-450 (FEI, Hillsboro, OR, USA) at 5– 10 kV. In the study was necessary to use the Nova Nano SEM-450 (FEI, Hillsboro, OR, USA) to show very small size and surface of sensilla (a pore system present or absent). 2.3. Image processing and morphometric measurement.
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Photographs and SEMs were observed and measured after being imported into Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA). 2.4. Terminology
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The terminology and classification of the labial sensilla is mainly based on the morphological criteria of Altner and Prillinger (1980), Zacharuk (1980), Brożek and Zettel (2014), and Brożek and
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Bourgoin (2013).
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3. Results 3. 1 Gross morphology of the mouthparts
The mouthparts of this species are similar to those of other heteropterans, consisting of a
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cone-shaped labrum, tubular segmented labium and stylet fascicle. The four labial segments differ in morphology and size (Table 1, Fig. 1A–1C). There are some sensilla symmetrically distributed on
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the labial surface including the apex. The labial tripartite apex is composed of two lateral lobes and a rostral lid. The lateral lobe has a sensory field bordered by a surrounding groove bearing 13 sensilla of
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different types (Fig. 6A–6C). The labrum is covered with numerous spike-like microtrichia (Fig. 2A and 2B). The mandibular stylet is covered with serrate ridges on the outer surface (Fig. 7C). Each maxillary stylet has a smooth external surface (Fig. 7F and 7G) and holds 2 internal grooves that together form a dorsal food canal (Fc) and ventral salivary canal (Sc), the right side being more developed (Fig. 7D and 7E). No obvious differences were noted in mouthpart structure and the length (p > 0.05) between females and males. The total length in females is 716.9 ± 32.6μm (n = 9), and for males is 702.5 ± 16.0μm (n = 4).
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3.1.1 Labrum The cone-shaped labrum, reaches half the length of the first labial segment (Fig. 1A and 2A). The basal part of labrum is wide but distal is elongated. Labrum morphologically divides into basilabrum (bl) strong sclerotized and distilabrum (dl) less sclerotized (Fig. 2A). Surface of the distilabrum is slightly an undulate. On ventral side of the distilabrum there is groove (gr), it keeps a proximal part of stylet fascicle (Sf) (Fig. 2B). Spike-like microtrichia (sm) are irregularly distributed throughout both surface labrum (dorsal and ventral) (Fig. 2A–2D). Four sensilla trichodea I (Fig. 2A and 2E) are also
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present. 3.1.2 Labium
The four labial segments are different in shape and size (Fig. 1A–1C). The first segment is stout, the base is broad and the end is narrowed. There is a bandlike dorsal plate (bdp) at the top that covers
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the joint between the two segments dorsally (Fig. 1B, 3A and 3B). Ventral membrane (vm) is well
distributed on the dorsal surface (Fig. 3A).
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developed between the first and the second segment (Fig. 1C and 3B). Sensilla trichodea II are
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The second segment is the longest, and the ends are relatively wide with a slight overlap in the middle (Fig. 4A and 4C). This segment contains three types of sensilla (sensilla basiconica I, sensilla trichodea II and sensilla campaniformia). There are three pairs of sensilla basiconica I at the base of
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the first sections (Fig. 3A and 4D). The dorsal, lateral and ventral surfaces bear some sensilla trichodea II (Fig. 4A–4C), and three pairs of sensilla campaniformia are irregularly arranged along
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the distal half of the second segment (Fig. 4A, 4F and 4G). The third labial segment is the shortest segment and is relatively broad (Fig. 4A–4C). In the
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proximal area of this segment on the anterior (dorsal) side there is small lobe (lo) arising on the left or right side and partly extended over the opposite side (Fig. 4A). The middle ventral part is slightly concave and two bands of undulated membrane (m1, m2) are visible (Fig. 4C). The distal end is convex ventrally (Fig. 4B and4C) and has few sensilla trichodea II on the dorsal, lateral and ventral surfaces (Fig. 4A–4C). The fourth, or distal, labial segment is tapered (Fig. 5A–5C). Several sensilla trichodea II are present laterally and ventrally. There is one pair of sensilla basiconica I at the base of the fourth
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segment. The labial tip is tripartite, distinctly divided into a rostral lid (rl) and two lateral lobes (Fig. 6A–6C). The rostral lid is formed by a small comb-like structure (Fig. 6A-6C). Two sensilla basiconica II (no 4, 9)(Fig. 6E), nine sensilla basiconica III (no 1-3, 5-8, 10-11), one flower-like sensillum (Fig. 6D) and one sensillum placodeum (Spl)(Fig. 6F) are found on the each of the sensory fields. The flower-like sensillum is present at the center of each lobe and an undulating, flower-like surface. A single sensillum placodeum is present at the side of each lobe.
3.1.3 Stylet fascicle
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The stylet fascicle is slender and composed of two separated mandibular stylets (Md) and two interlocked maxillary stylets (Mx) (Fig. 7A and 7B). The mandibular stylets (Md) are adpressed laterally to the maxillary stylets (Mx). The mandibular stylets have an internal concavity allowing them to precisely cover the maxillary stylets. The mandibular stylet tips have about 30 pairs of lateral
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small teeth (Sr), which may help in penetrating leaf tissue. Each maxillary stylet has 2 grooves that
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together form a dorsal food canal (Fc) and ventral salivary canal (Sc), the right side being more developed (Fig. 7D and 7E). The ends of the external surfaces of the maxillary stylets are smooth but
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on the inner ridges there are five small, slightly recurved teeth (Fig. 7D). The end of the right maxillary stylet is lancet-shaped (Fig. 7D–F) in contrast to the left stylet, which is blunt and apically slightly divided (Fig. 7E, G).
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The cross-sections of this lace bug species (Fig. 8) show that the two mandibular stylets are mirror symmetric and the two maxillary stylets are asymmetric. The mandibular and maxillary stylets
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are connected to each other through a T-shaped process. The two maxillary stylets have dorsal, middle and ventral inner locking systems (Fig. 8). The dorsal lock has two hooked process and two straight
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process. The middle lock has one T-shaped process and two hooked processes. The ventral lock has three hooked process and one straight process (Fig. 8). 3.2. Type and distribution of the mouthpart sensilla Under scanning electron microscopy five types of sensilla were observed on the mouthparts of adult males and females: sensilla basiconica (including three subtypes: Sb I, Sb II and Sb III), s. trichodea (including two subtypes: St I and St II), flower-like sensillum, s. campaniformia (Sca) and s.
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placodea (Spl), respectively. No consistent difference was found in abundance and distribution of sensilla between the male and female adults. 3.2.1 Sensilla trichodea Sensilla trichodea I (St I) are very long and slightly curved. The base of the sensilla have a flexible socket, the surface with a slight vertical groove and the tip is narrow and rounded (Fig. 2A and 2E). The length of St I is 58.13–90.79 μm, and the basal diameter is 2.23–2.57 μm. This type of sensillum was found on the labrum (distilabrum).
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Sensilla trichodea II (St II) are short and thick. The base of the sensilla has flexible socket and a slight vertical grooved surface (Fig. 3A, 3C, 3D, 4F, 5A–C). The length of St II is 11.42-19.47 μm, and the basal diameter is 1.43-2.02 μm. This type of sensillum occurs on the labium.
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3.2.2 Sensilla basiconica
Sensilla basiconica I (Sb I) are short, small and straight. The surfaces of sensilla are smooth and
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have a rounded tip (Fig. 3A, 4D and 4E). Sb I is 5.19–7.75 μm, and the basal diameter is 1.85–2.02
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μm. They are distributed between the first and second labial segments. Two sensilla basiconica II (Sb II) are present at the center of the apical sensory field (no 4, 9), and with a terminal pore and sit in a tightly fitting socket (Fig. 6E). Sb II is 2.58–5.36 μm, and the
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basal diameter is 1.18–1.42 μm.
Nine sensilla basiconica III (Sb III) are present at the center of the distal sensory field. Sb III is
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5.12–8.15 μm, and the basal diameter is 0.84-1.24. They are short, small, straight and have a smooth
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surface, a rounded tip and sit within a tightly fitting socket (Fig. 6C, no 1–3, 5–7, 10–11). 3.2.3 Sensilla campaniformia These are flat and oval shaped discs. Three pairs of sensilla campaniformia are distributed on the
second labial segment (Fig. 4F and 4G). The basal diameter is 4.63–6.13 μm. 3.2.4 Flower-like sensilla The flower-like sensillum is located in the middle of the lateral lobes of the labial tip (Fig. 6B). This sensillum is recognized as “flower-like sensillum” that consists of folds (fo) with very small
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pegs (pe) (Fig. 6D). Both structures are aporous, but probably the pegs have sensory function. The diameter of the undulated surface is 3.6–4.64μm. 3.2.5 Sensilla placodea These are oval shaped, elevated above the surface, with multiporous walls. This type of sensillum (Fig. 6F) occurs on each lateral lobe of the labial tip. The basal diameter is 3.16–3.44 μm. 4. Discussion Morphological studies of mouthpart fine structure reveal important information related to
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feeding behavior as well as evolutionary relationships. Scanning electron microscopy was used in this study to document previously understudied details of the mouthparts of Tingidae (Cimicomorpha) based on the economically important species S. nashi. Comparison of mouthpart structures of this
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species to that of other Heteroptera may help to explain modifications of these structures and their function.
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Labrum in most studied is very briefly discussed. According to Štys (1969) in Cimicomorpha types of the labrum have been recognized. One short, wide, extended labrum was found in few
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families. Other type of labrum as spiniform elongated and divided in basilabrum and distilabrum (often less sclerotized) was indicated e.g. in some Reduviidae. Latter type is similar to the tingid
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labrum of Stephanitis nashi.
The labium of tingids is four-segmented (Stehlik, 1997) as in most other heteropteran bugs (Schuh and Slater, 1995). Moreover, three specific labial characters were found in S. nashi: a dorsal
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bandlike plate and a ventral membrane are well developed between the first and the second segment,
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which affects the bend of labium during feeding (Fig. 3B). A similar bending mechanism is present in Pyrrhocoris sibiricus (Pyrrhocoridae) (Wang and Dai, 2017). The third segment in the ventral part is slightly sclerotized and mostly formed by a flexible/undulated membrane which influences flexibility during feeding. The long, stiff second segment holds the stylets and probably controls bending of the short third and fourth segments. The dorsal lobe of the third segment holds the stylets within the labial groove during feeding (Fig. 1A). This lobe is characteristic of Stephanitis nashi and has not been reported in other heteropterans.
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Labial
sensilla
and
their
functions
as
chemoreceptors,
mechanoreceptors
and
thermo-hygroreceptors were previously described in various hemipteran taxa (Brożek and Bourgoin, 2013; Rani and Madhavendra, 1995, 2005; Foster et al., 1983; Backus and McLean, 1982; Zhao et al., 2010). In S. nashi, two symmetrical fields on the labial tips are equipped with sensilla of three morphological types: two sensilla basiconica II (SbII), nine sensilla basiconica III (Sb III) and one multiporous sensillum placodeum (Spl); another flower-like sensillum is also present on each field. Variation in shape between two types of sensilla (Sb II and Sb III) in S. nashi may reflect same chemosensory functions. The single pore characterizing these sensilla is either terminal or
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sub-terminal suggesting a gustatory (Sb II) or contact-chemosensory function (Sb III gustative and touch). The latter are placed around a center of the labial tip and they are slightly longer than Sb II. Gustatory sensilla are mostly bimodal, comprising chemo- and mechanosensitive receptor cells
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(Zacharuk, 1980; Altner and Prillinger, 1980; Steinbrecht, 1992; Chapman, 1998; Shields, 2010).
Three pairs of sensilla campaniformia in S. nashi are distributed at the distal part of the dorsal
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surface of the second labial segment, they may function as proprioceptors responding to the stresses connected with the movement of the labium. Similar mechanosensilla were identified as
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proprioceptors in Peiratinae (Reduviidae) (Catalá, 1994), in the seed-bug, Pyrrhocoris sibiricus Kuschakevich (Wang and Dai, 2017) in phytophagous Pentatominae and in predatory Asopinae
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(Pentatomidae) (Parveen et al., 2015). It is common for proprioceptive sensilla campaniformia (Sca) to occur on mouthparts, antennae, bases of wings, halteres, legs, and eyes (Schneider, 1964; Bromley and Dunn, 1980; Wang et al., 2013; Zhang et al., 2016).
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The gustatory sensilla are generally abundant reflecting their important role in host location and
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evaluation. These sensilla are usually perpendicular to the labial tip and this position allows them easily to contact the substrate. Distribution and number of gustatory sensilla (9+2) in S. nashi resemble those of phytophagous mirids (e.g., 11 peg/basiconic sensilla in Lygus rugulipennis and L. lineolaris) (Avé et al., 1978; Hatfield and Frazier, 1980; Romani et al., 2005). S. nashi shares a similar pattern of gustatory sensilla with other plant bugs including the pentatomid Nezara viridula L. (Rani and Madhavendra, 1995) and the chinch bug Blissu sleucopterus leucopterus (Say) (Baker et al., 2008). However, the pattern is not stable and in some other plant bugs Dysdercus intermedius, D. fulvoniger and D. koenigii there are only 10 gustatory sensilla (Gaffal, 1981) while 9 such sensilla
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have been identified in the alydid Riptortus pedestris F. and 8 in the lygaeid Elasmolomus sordidus (F.) (Rani and Madhavendra, 2005). There is a significant difference in shape of the gustatory sensilla between S. nashi and predatory triatomine and peiratine assassin bugs (Reduviidae) (Brożek and Chłond, 2010; Catalá, 1996; Rosa et al., 1999). The latter have short uniporous peg sensilla corresponding to the sensilla basiconica of S. nashi, but reduviids also have dome-elongated uniporous sensilla (UDES) placed more subapically in relation to the peg sensilla. Similarly to some other cimicomorphans (Brożek and Chłond, 2010), the labial olfactory system in the studied species includes one multiparous sensillum. We hypothesize that this type of sensillum
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functions as an olfactory chemoreceptor, which may sense plant volatiles prior to feeding. It seems that in S. nashi (Tingidae) a typical sensillum placodeum may correspond to the sensillum basiconicum with a perforated wall in other phytophagous species, e.g., Dysdercus spp. and
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Pyrrhocoris sibiricus (Brożek and Zettel, 2014; Brożek and Bourgoin, 2013; Wang and Dai, 2017; Gaffal, 1981; Peregrine, 1972).
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An aporous flower-like sensillum is distributed at the center of each lobe. Aporous sensilla (e.g. sensilla basiconica and pegs) are associated with mechanoreception such as touch, thermo-, and
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hygroreception (McIver 1985). Aporous flower-like sensilla may be thermo-hygroreceptive in S. nashi. The fine structure of thermo-hygroreceptive aporous sensilla is conservative across insect
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orders (Altner et al., 1983; McIver, 1973; McIver and Siemicki, 1976; Steinbrecht and Müller, 1961; Yokohari, 1981). In S. nashi, aporous flower-like sensilla are located in the center of each lateral lobe and we provide the first detailed description.
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In S. nashi, the mandibular tips have at least 30 shallow serrations which extend somewhat
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transversely as also reported in A. carinata (Tingidae) (Cobben, 1978). The serrations are fewer in number (11) but deeper in D. nebulosus and other species of mirids (Boyd et al., 2002). Boyd et al. (2002) pointed out that the mandibular stylets of D. nebulosus are typical for Miridae regardless of whether they are phytophagous or predatory. The serrations on the mandibular stylet tip may be used to fix the stylets in host tissues (Cobben, 1978; Cohen, 2000; Wheeler, 2001). Different patterns of weak mandible serration (blunt and wide teeth) are present in other phytophagous species of Coreidae and Lygaeidae (Cobben, 1978; Gaffal, 1981; Cohen 1990; Depieri and Panizzi, 2010). These bugs produce a salivary flange that may act as a fulcrum for probe movement (Cohen, 1998, 2000).
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In this species the maxillary stylets (Mx) are smooth on the outer side but on the inner side are equipped with five small teeth directed away from the head (7D). The end of the right maxilla is lancet-shaped in contrast to a slightly divided end of the left maxilla. Additionally, the mandibular stylet tips are serrated with 30 pairs of wide and small teeth (Sr). Similar structures occur in another tingid species, Acalypta carinata, in which there are also five rudimentary teeth on the inner side of the maxillary stylet and several shallow indentations on the mandibular ends (Cobben, 1978). Maxillary stylets similar to S. nashi (Tingidae) were also observed in predatory species of mirids. Deraeocoris nebulosus and D. olivaceous have six teeth on the inner surface and D. nigritulus has
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seven teeth pointed distad (Cobben, 1978; Boyd et al., 2002; Boyd, 2003). The teeth in Deraeocoris spp. are slightly longer than those in S. nashi but the significance of such slight differences remain to be revealed by comparative study of additional species. The structure of the maxillary stylets of
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Miridae has been suggested to represent an intermediate condition between phytophagous and predatory types (Wheeler, 2001). Some observations suggest that the right maxillary stylets of
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predatory Heteroptera/Cimicomorpha (e.g., Nabidae, Anthocoridae, Reduviidae) are more deeply serrated and have more numerous barbs than those in phytophagous species in which the apices of the
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maxillae are only slightly serrated on the inner side (Tingidae, Lygaeidae, Pentatomidae, Pyrrhocoridae) (Khan, 1972; Cobben, 1978; Cohen, 1990; Wang and Dai, 2017). The orientation of
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the teeth on the maxillary stylets is also different. The maxillae of zoophagous heteropterans are armed with backward-curved teeth which better anchor the stylets in the tissues of struggling prey
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(Cobben, 1978; Cohen, 1996). 5. Conclusions
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Our detailed study of mouthpart fine structure in S. nashi represents the first comprehensive description of sensory structures of lace bug mouthparts. S. nashi (Tingidae) mouthparts show modifications similar to those found in phytophagous and zoophagous mirids. Morphological evidence indicates that many sensilla at the labial tip have gustatory and olfactory functions, consistent with their role in host plant selection.
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Acknowledgments: We are grateful to Chris Dietrich who reviewed the manuscript and offered several valuable suggestions for improvement. We also thank the Life Science Research Core Services of Northwest A&F University for providing scanning electron microscope. This project was supported by the National Natural Science Foundation of China (Nos. 31772514, 31572306, 31272343).
Conflicts of Interest: The authors declare no conflict of interest.
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Figure legends
Figure 1. Scanning electron micrographs of the head of Stephanitis nashi. A. Dorsal view; B. Lateral view; C. Ventral view showing four-segmented labium (I–IV); Sf, stylet fascicle; Lm, labrum; Lb,
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labium; bdp, bandlike dorsal plate; vm,ventral membrane.
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Figure 2. SEM of labrum of Stephanitis nashi. A. Dorsal view of labrum (bl-basilabrum),
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dl-distilabrum), sensilla trichodea I (St I) on distilabrum; B. Ventral view of labrum (distilabrum); C. Enlarged view of tip of labrum; D. Enlarged view of box in (B); E.
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Enlarged view of sensillum trichodeum I (St I); sm, spike-like microtrichia; gr, groove; Sf,
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stylet fascicle.
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Figure 3. SEM of first labial segment of Stephanitis nashi. A. Lateral view of the first labial
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segment; B. Ventral view; C. Enlarged view of first labial segment; D. Enlarged view of
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sensillum trichodeum II (St II); bdp, bandlike dorsal plate; vm,ventral membrane.
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Figure 4. SEM of the second and third labial segment.A. Dorsal view; B. Lateral view; C. Ventral view; D. Enlarged view of box in (A), showing the base of the second segment; E. Enlarged view of sensillum basiconicum I (Sb I). F. Enlarged view of box in (A), showing the surface of the second segment; G. Enlarged view of sensillum campaniformium (Sca);
lo, lobe; m, membrane.
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Figure 5. SEM of the fourth labial segment of Stephanitis nashi. A. Dorsal view; B. Lateral
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view; C. Ventral view.
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Figure 6. SEM the tip of labium of Stephanitis nashi. A. Ventrolateral view; B. Apical view showing flower-like sensillum (black circles); C. Lateral view; D. Enlarged view of
flower-like sensillum; E. Enlarged view of sensillum basiconicum II-4 (SbII-4); F. Sensillum placodeum (Spl) (dome-shaped multiporous sensillum). rl, rostral lid; p, pore; fo, fold; pe, peg.
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Figure 7. SEM images of stylets of Stephanitis nashi. A. Enlarged ventral view of stylet fascicle apex; B. Enlarged ventral view of stylet fascicle showing mandibular stylets (Md) and maxillary stylets (Mx); C. Tip of mandibular stylets (Md) showing serrate ridge (Sr); D. Tip of inner surface of right maxillary stylet (RMx); E. Tip of the inner surface of left maxillary stylet (LMx); F. Enlarged view of tip of right maxillary stylet (RMx); G. Enlarged view of tip of left maxillary stylet (LMx); Fc, food canal; Sc, salivary canal.
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Figure 8. Diagram of cross-section of stylet fascicle of Stephanitis nashi. A, Hooked; A’,
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Straight; B, Straight; B’, Hooked; C, T-shaped; C’, Hooked; D, Hooked; D’, Hooked; E, Straight; E’, Hooked; CN, nerve canal; Fc, food canal; LMd, left mandibular stylet; LMx,
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left maxillary stylet; LPr, left process of the maxilla; RMd, right mandibular stylet; RMx,
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right maxillary stylet; RPr, right process of the maxilla; Sc, salivary canal.
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Table 1. Measurements of labrum and labium (mean ± SD) obtained from scanning electron microscopy. N = sample size. Lm, labrum; Lb, labium; Lb-sg1, first segment of labium; Lb-sg2, second segment of labium; Lb-sg3, third segment of labium; Lb-sg4, fourth segment of labium. Gender
Position
Length (μm)
Width (μm)
Male
Lb
702.5 ± 16.0
Female
Lm
193.2 ± 17.3
Lb
716.9 ± 32.6
Lb-sg1
213.8 ± 17.1
80.7 ± 4.0
9
Lb-sg2
269.0 ± 15.1
46.5 ± 2.7
9
Lb-sg3
114.3 ± 5.8
47.8 ± 6.2
9
Lb-sg4
119.8 ± 4.9
42.3 ± 4.1
9
4 74.4 ± 3.4
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N