Structure & Development 41 (2012) 79e86

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Arthropod Structure & Development

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Morphological description of the mouthparts of the Asian citrus psyllid, citri Kuwayama (: Psyllidae)

E. Garzo a, J.P. Bonani b, J.R.S. Lopes b, A. Fereres a,* a Departamento de Protección Vegetal, Instituto de Ciencias Agrarias (ICA, CSIC), C/Serrano 115 dpdo, 28006 Madrid, Spain b Departamento de Entomologia e Acarologia, ESALQ/Universidade de São Paulo, CP. 9, Piracicaba, SP 13418-900, Brazil article info abstract

Article history: Scanning (SEM) and transmission (TEM) electron microscopy were used to elucidate the morphology of Received 14 April 2011 the rostrum, as well as the mandibular and maxillary stylets of the psyllid , vector of Accepted 24 July 2011 phloem-inhabiting bacteria associated with citrus huanglongbing (HLB) disease. D. citri has a cone- shaped rostrum that extends behind the pair of prothoracic coxae. The stylet bundle comprises a pair Keywords: of mandibular (Md) and maxillary (Mx) stylets with a mean length of 513.3 mm; when retracted, their Diaphorina citri proximal portions form a loop and are stored in the crumena (Cr). Serial cross-sections of the rostrum Electron microscopy revealed that the mandibles are always projected in front of the maxillary stylets. The two maxillary Mouthparts stylets form the food and salivary canals, with diameters of 0.9 mm and 0.4 mm respectively. These two HLB m Ca. canals merge at the end of the stylets forming a common duct with a length of 4.3 m and a mean Transmission diameter of 0.9 mm. The acrostyle, a distinct anatomical structure present in the common duct of Disease maxillary stylets, was not observed by TEM in the ultrathin cross-sections of the common duct (CD) of D. citri. This study provides new information on D. citri mouthparts that may help to understand the feeding behaviour of this important vector of HLB-associated bacteria. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Rani and Madhavendra,1995; Feeman et al., 2000, 2001; Boyd et al., 2002; Boyd, 2003; Wiesenborn, 2004; Anderson et al., 2006; Uzest The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: et al., 2010; Zhao et al., 2010). Piercing-and-sucking mouthparts Psyllidae), is an invasive species that has emerged as the most show similar structures in the different hemipteran families. The important pest of world citriculture. It was discovered in Taiwan in mouthparts are composed of three different structures: a short and 1907 and is presently distributed across several countries of South triangular labrum, which covers the base of the stylet bundle; the Asia, regions of the Middle East, Réunion and Mauritius islands, the labium, which is a segmented and tubular organ with a complex Southern region of the United States, Central America, the Carib- musculature that contracts and shortens during insertion of the bean and South America (Halbert and Manjunath, 2004). D. citri is stylet into the plant tissue; and the stylet bundle inserted in vector of the phloem-inhabiting bacterium Candidatus Liberibacter a groove located along the length of the inner surface of the labium. asiaticus, which is associated with Asian ‘huanglongbing’ (HLB), The stylet bundle has two external mandibular stylets that commonly referred to as citrus greening disease in English- surround and protect two inner maxillary stylets (Pollard, 1973; speaking countries (Capoor et al., 1967; Bové, 2006). HLB is Rosell et al., 1995; Leopold et al., 2003; Zhao et al., 2010). a devastating disease that reduces fruit yield and quality and can The mouthparts of (Hemiptera: ) have been kill or severely debilitate citrus trees. Therefore, D. citri may be one studied in detail by several authors because of the important role of of the most serious and economically important pests of aphids as virus vectors. These authors described the ultrastructure citrus where HLB occurs (Gottwald et al., 2007). of different aphid species using electron microscopy techniques Abundant information about the ultrastructure morphology of (TEM and SEM) in order to obtain information about the sensory the mouthparts of Hemiptera, based on light, transmission (TEM) neurones which innervate the mandibular stylets (Wensler, 1974), and scanning (SEM) electron microscopy is available (Pollard, 1973; the characteristic interlocking of the maxillary stylets, where the food and salivary canals are separated along the length of the stylets, and how both canals are fused into one common duct in the * Corresponding author. Tel.: þ34 91 7452500; fax: þ34 91 6540800. last few distal microns (Forbes, 1969; Mittler, 1957). Furthermore, E-mail address: [email protected] (A. Fereres). some authors described the labial tip receptors and speculated

1467-8039/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2011.07.005 80 E. Garzo et al. / Arthropod Structure & Development 41 (2012) 79e86 about the possible role they played (Wensler, 1977; Tjallingii, 1978), binocular microscope using very fine dissecting needles obtained and others have studied the movement and penetration of aphid by sharpening tungsten wire electrolytically (Brady, 1965). The stylets (Weber, 1930; Bradley, 1962; Evert et al., 1973). The most samples were carefully mounted on a gold specimen holder using recent study of the ultrastructure of the aphid mouthparts was OCT compound (Tissue-Tek, Sakura, USA). The specimen holder was made by Uzest et al. (2010), who described the ultrastructure of the then plunged into liquid nitrogen slush at approximately 210 C. tips of aphid maxillary stylets and demonstrated the existence of The sample was immediately transferred in a closed-air transfer a distinct and well-defined anatomical structure, named the device onto the cryostage of a CT1500 Oxford attached to a Zeiss "acrostyle", which is a swollen part of the cuticle present in the DSM960 scanning electron microscope. Sublimation of surface frost common duct of all aphid species observed. was performed at 90 C for 2 min before sputter-coating the Compared with aphids, there is less information available about sample with gold for 2 min at 10 mA. After sputter-coating, the the morphology and fine structure of the mouthparts of sample was imaged at 15 kV in the precooled chamber at 150 C. (Hemiptera: Cicadellidae), another important group of vectors of plant pathogens. There is some published information on how the 2.3. Transmission electron microscope (TEM) structure of Homalodisca coagulata (Say) mouthparts relates to the function of locating the xylem tissue within the plant host (Leopold The were fixed in 3% glutaraldehyde, 0.1 M sodium et al., 2003). Backus and McLean (1982, 1983) studied the ultra- cacodylate buffer pH 7, 1.8% saccharose, 0.001 M CaCl2, and after structure of the sensilla and other structures within the stylets and 24 h the insect mouthparts, with the clypeus, labium and stylets, the precibarium of Macrosteles fascifrons (Stål) and other leafhop- were removed using two fine tungsten dissecting needles under pers. Backus (1985) presents a review on anatomy of mouthparts, binocular microscopy. foregut and associated sensory organs of leafhoppers (Cicadellidae) The samples were washed and left for 1 h in the sodium caco- and (Fulgoroidea). More recently, Zhao et al. (2010) dylate buffer. In order to obtain transversal sections the samples has used microscopic analysis to study the reputed functions of were embedded in 1% agar to simplify specimen orientation during the mouthparts of the Psammotettix striatus (L.). inclusion in the resin. After washing in distilled water, the samples However, within the Psyllidae, all studies have been restricted to were post-fixed in 1% osmium tetroxide for 2 h and the block with the genus . Some anatomical studies of the head and the tissue was left for 1 h in 0.5% uranyl acetate at 4 C. The samples feeding structures of the apple , Cacopsylla (Psylla) mali were dehydrated in ethanol series, infiltrated and embedded in (Schmidberger), have been made by Grove (1920). Forbes (1972) Spurr-type resin. Ultrathin sections (70e80 nm) of the labium and observed dendrites in the mandibular stylets of the pear psylla, stylet bundle were cut using an ultramicrotome and placed on Cacopsylla (Psylla) pyricola (Foerster) and Pollard (1970) examined pioloform-coated copper grids (50 mesh) and stained with 2% stylet movement in the apple psyllid. Furthermore, Ullman and aqueous uranyl acetate (Reynolds, 1963). The samples were McLean (1986) studied the anterior food canal of the pear psylla. examined under a STEM LEO EM910 transmission microscope Recently, detailed information on the probing and feeding (Oberkochen, Germany), equipped with a digital Gatan Bioscan 792 behaviour of D. citri has been collected to understand the trans- camera (Pleasanton, CA, USA). mission process of Ca L. asiaticus (Bonani et al., 2010). However, no studies on the ultrastructure of D. citri mouthparts have been made 3. Results to date. Hemipteran mouthparts play an essential role in the transmission of plant pathogenic bacteria and viruses, and there- The mouthparts of D. citri are similar to those of other piercing- fore, a detailed study of the structure and function of D. citri may sucking insect pests. In Fig. 1D, the different structures that provide new insights into the behavioural events associated with constitute the mouthparts can be seen: the clypeus (Cp), which the transmission of Ca L. asiaticus and related bacteria by its vector. contains the cibarium dilator muscles (CbDm), the cibarium (Cb), The focus of this study was to examine the ultrastructure of followed by the labium or rostrum (Lb). It is important to note that D. citri mouthparts, including the labium and the mandibular, when the stylets are retracted, the distal part is kept inside the maxillary stylets and the common duct, using transmission (TEM) labium and the proximal part returns in a loop so that the stylets and scanning electron microscopy (SEM). are covered by a membrane called the crumena (Cr).

2. Materials and methods 3.1. Labium

2.1. Insect rearing The labium of D. citri has a mean length of 233.2 4.7 mm (n ¼ 10, range 209.1e254.5), and lies protected between the All the insects used in the study were 7e14 day old adults prothoracic coxae of the first and second pair of legs (Fig. 1A and C). obtained from a laboratory colony originally collected on citrus in This structure, which holds two mandibular and two maxillary Piracicaba, SP, Brazil, in 2007 and maintained on jasmine, stylets, is subdivided into three segments (Fig. 1B). The two distal Murraya paniculata (L.) Jack (Rutaceae), in a controlled- segments of the labium (Lb-sg1 and Lb-sg2) are exposed to the environment room (26 2 C; 70% RH and L14:D10 photope- outside, leaving the third proximal segment (Lb-sg3) hidden riod), as described by Bonani et al. (2010). Voucher specimens are between the prothoracic coxae (Fig. 1C). The three segments differ deposited at ESALQ Entomological Museum, Department of Ento- in size and morphology (Table 1; Fig. 1B); the Lb-sg1 segment is mology and Acarology, ESALQ/University of São Paulo, Piracicaba, narrower and thinner than the other two segments, has a conical SP, Brazil. shape and is narrower on the extreme distal portion. Three measurements of the width of each Lb-sg1 segment were taken: 2.2. Low temperature scanning electron microscope (LTSEM) one proximal, one central and one distal (Table 1; Fig. 2B). The Lb- sg2 and Lb-sg3 segments have different length but the same width Adults of D. citri were fixed in 70% ethanol and three different (Table 1). types of samples were prepared: (1) the whole insect; (2) the At the end of the distal extremity (Lb-sg1), a group of labium; (3) the stylet bundle. The labium and the stylet bundle with symmetrically bilateral sensilla can be seen, with four sensilla on the clypeus of the dead insect were freed under a dissecting each side of the rostrum opening (Fig. 1E). These sensilla have E. Garzo et al. / Arthropod Structure & Development 41 (2012) 79e86 81

Fig. 1. Ultrastructural study of the mouthparts of Diaphorina citri. Scanning electron microscopy (SEM) of the (A) Ventral view of an adult, where the labium is protected between the prothoracic coxae. (BeC) Detail of labium. (D) Lateral view of the mouthparts. (E) Detail, distal extremity of labium showing apical sensilla. Cibarium (Cb); cibarium dilator muscles (CbDm); clypeus (Cp); crumena (Cr); coxa (Cx); labium (Lb); segments of the labium (Lb-sg1, 2, 3); mandibular stylets (Md); sensilla (Sl); stylets (Sty). Bars represent in A and D: 200 mm, in B, 100m,inC:50mm and E: 10 mm. 82 E. Garzo et al. / Arthropod Structure & Development 41 (2012) 79e86 a mean length of 5.2 0.3 mm(n ¼ 8, range 4.2e6.4) and a mean maxillae are hold together by an interlocking tongue and groove diameter of 1.3 0.07 mm(n ¼ 8, range 1.1e1.6). They are involved system (Fig. 3GeH). The two internal grooves form the food (FdC) in host selection and are part of the insect’s sensorial structures. and salivary canals (SC) when the maxillae are connected to each other. 3.2. Stylet bundle The two maxillae (Mx) can be seen in Fig. 3D, but in only one of them it is possible to visualize the grooves in their entirety, in particular the two central ones, which represent the beginning of The stylet bundle is like a needle composed by a pair of 1 1 mandibular and maxillary stylets. It has a mean length of the food (FdC ) and salivary (SC ) canals. In the next section 513.3 13.4 mm(n ¼ 9, range 460.7e561.7) measured from the towards the proximal portion of the labium (Fig. 3E), the beginning clypeus (Cp) to the distal extremity (Fig. 2A). of the junction of the maxillae can be seen, demarcating the food The mandibles are simple structures with few morphological (FdC) and salivary (SC) canals. These canals become more evident variations, convex externally and concave internally. The distal with the approach of the second maxillary stylet (Fig. 3F). With the extremity of the convex region is composed of ten “grooves” in each complete junction of the two maxillary stylets (Fig. 3G), the canals mandible (Fig. 2B), which probably help stylet penetration of the are completely separated and independent. Fig. 3H represents the tissues; whereas the internal concave region has the function of most proximal section of the labium, showing no further stylet holding the maxillary stylets. Fig. 2CeE show an electron micro- morphological differentiation. scope scan of the internal region of the maxillary stylets of the Asian citrus psyllid. In Fig. 2C, we can observe the clear separation 4. Discussion of the food and salivary canals and in Fig. 2DeE, the concave or internal region of the next maxillary stylet (Mx). When the The morphological structures of the mouth apparatus of D. citri maxillary stylets are united by the hinges, located to the edges of appear to be similar as those in aphids and leafhoppers, and like the maxilla, the following ducts are formed: food (FdC), salivary those also described by Ullman and Mclean (1986) for the pear (SC) and the common duct (CD) (Fig. 2E). The common duct (CD) of psylla, P. pyricola. The Asian citrus psyllid has a stylet bundle D. citri has a mean length of 4.3 0.04 mm(n ¼ 5, range 4.2e4.4) comprised by a pair of mandibular stylets located externally, which and a mean diameter of 0.9 0.02 mm(n ¼ 5, range 0.8e0.9). The house a pair of maxillary stylets, as observed in aphids (Pollard, measurements of the diameters of the food (FdC) and salivary (SC) 1973) and other psyllids, including P. mali (Grove, 1920; Ullman canals were taken at a distance of about 15 mm from the most distal and McLean, 1986) and P. pyricola (Forbes, 1972). In D. citri, the part of the maxillary stylets, and measure 0.9 0.04 mm(n ¼ 5, stylet bundle is housed within a rostrum or labium located between range 0.8e1.0) and 0.4 0.04 mm(n ¼ 5, range 0.3e0.5), respec- the first and second pair of legs (Fig. 1A and C). Unlike the insect tively. Using SEM, we were able to observe a distinctive surface structure described for aphids (Pollard, 1973), the rostrum of D. citri appearing as a slight swelling of the cuticle in part of the common is not completely housed in the ventral thoracic region and is duct of D. citri (Fig. 2D and E), somehow similar to that described for projected backwards between the coxae of the first pair of legs aphids and named the “acrostyle” by Uzest et al. (2010). This when the insect is not feeding; instead, D. citri always holds its distinctive ultrastructure was observed in both the left (Mx1) and rostrum in a vertical position (feeding position). This is probably right (Mx2) maxillary stylets of D. citri. However, when looking due to a small retraction of the rostrum when resting, and as soon a series of ultrathin cross-sections of the common duct (CD) as it starts feeding the insect assumes the characteristic feeding observed by TEM (Fig. 2 series F1-7), the presence of an acrostyle- position, forming a 45 angle in relation to the leaf surface, like structure in D. citri was not confirmed. favouring contact of the bucal apparatus with the substrate. Due to The serial transverse sections in Fig. 3AeH show the internal this typical morphology, D. citri has a short rostrum with a mean aspect and distribution of the labial structures of D. citri and are length of 233.2 4.7 mm, which adjusts well to the feeding habits of representative along their entire length, from the distal to the this insect. When D. citri is not feeding, the stylets are housed in proximal portions. We studied the configuration of the stylets a bag called the crumena, as seen in Fig. 1D, which is formed by an inside the labium following the section sequence (Fig. 3AeH). In invagination of the insect’s body wall on the labium base (Forbes, Fig. 3AeB, we only observed the mandibles (Md) with the neural 1969). This structure is characteristic of insects with very long ducts, which were always projecting in front of the maxillae (Mx). and filamentous stylets, and is present in some hemipteran fami- This is because in many Hemiptera, the mandibles are also lies, such as Aleyrodidae, Coccidae and Psyllidae (Pollard, 1970, responsible for opening up plant tissues, not only externally on the 1973; Maranhão, 1978). epidermis but also internally (mesophyll cells and vessels). The At the distal extremity of the labium, D. citri has four pairs of presence of one of the maxillae (Mx), housed by the mandibles bilaterally symmetrical sensilla (Fig. 1E). These structures probably (Md), and the beginning of the common duct (CD) can be observed constitute part of the psyllid’s sensory system for recognizing hosts in Fig. 3C. Morphologically, in contrast to the mandibles, the as shown for aphids and whiteflies (Tjallingii, 1978; Walker and maxillae are not bilaterally symmetrical, and are more complex. Gordh, 2001), but it is not known if they have a chemosensory or Externally, the maxillary stylets are equal (convex), but internally mechano-chemosensory function in D. citri. Eight or seven pairs of they have four longitudinal grooves that differ in shape. The two apical labial sensilla were reported for aphids (Tjallingii, 1978) and

Table 1 Mean SE values (mm) of the length and width of the three segments of Diaphorina citri labium. Range values are given in parentheses. Sample size is 10.

Labium Length Width

Mean SE Proximal Central Distal

Mean SE Mean SE Mean SE Lb-sg1 66.4 1.0 (60.5e72.7) 40.5 0.7 (38.0e43.8) 32.5 0.7 (30.3e36.9) 16.9 0.5 (15.1e20.7) Lb-sg2 71.6 2.8 (54.3e84.5) 57.5 1.6 (48.3e62.7) Lb-sg3 95.1 2.1 (83.1e103.1) E. Garzo et al. / Arthropod Structure & Development 41 (2012) 79e86 83

Fig. 2. Scanning electron microscopy (SEM) of the stylets of Diaphorina citri. (A) View of the stylets next to the clypeus; (B) External detail of one of the mandibular stylets; (C) Detail of the food and salivary canals; (DeE) Distal extremity of maxillary stylets, with detail of the common duct. A distinctive ultrastructure, similar to the acrostyle of aphids, is observed along the bottom of the common duct (indicated by black-white arrow heads). A series of cross-sections of the tip of the maxillary stylets (F1-F7) are observed by transmission electron microscopy (TEM). The acrostyle is not observed in the common duct of D. citri. Clypeus (Cp); common duct (CD); food canal (FdC); grooves (Go); left maxillary stylet (Mx1); mandibular stylet (Md); maxillary stylet (Mx); right maxillary stylet (Mx2); salivary canal (SC); stylets (Styl). Bars represent in A: 200 mm, in B: 10 mm, in C and D: 5 mm, in E: 2 mm and in F1eF7: 0.2 mm. 84 E. Garzo et al. / Arthropod Structure & Development 41 (2012) 79e86 whiteflies (Walker and Gordh, 2001), respectively; but for other perforating plant tissues during stylet movement. They are also psyllids we found no information on the number of these sensilla. present in aphids and Forbes (1969) described more than 10 of Aphid labial sensilla are not only more numerous but also longer these teeth-like structures in M. persicae. (around 10 mm in length) than those observed in D. citri, whose Transmission Electron Microscope (TEM) sections of D. citri mean length is 5.2 mm(Fig. 1E). show stylet configuration from the distal extremity to the proximal The stylets of aphid vectors have been studied in detail because portion of the rostrum (Fig. 3AeH). The pair of mandibular stylets is they are intimately linked to plant virus transmission mechanisms advanced further ahead than the maxillary stylets (Fig. 3A, B), and (van Hoof, 1957,1958; Forbes, 1969; Pollard,1973). The mean length this configuration was always seen in the samples of D. citri of the stylet bundle of D. citri (513.3 mm) is slightly longer than that examined. This is the case when other Hemiptera begin stylet the green peach aphid, Myzus persicae (Sulzer), which measures penetration into the plant tissues (Weber, 1930). Pollard (1970) 502 mm for winged and 492 mm for apterae morphs (Forbes, 1969), studied this stylet movement in detail for P. mali. The beginning and shorter than the stylet bundle of the rice brown , of the common duct of D. citri arises from the first maxilla (Fig. 3C), Nilaparvata lugens (Stål), which measures 650 mm(Foster et al., and is formed by the junction of the food and salivary canals 1983). Ullman and McLean (1986) described the morphological (Forbes, 1969; Tjallingii, 1978); this common duct is 4.3 mm long. characteristics of the bucal apparatus of psyllids as being inter- Previous studies with other Hemiptera reported only the length of mediate between aphids () and planthoppers or the food and salivary canals (Forbes, 1969; Forbes and Mullick, leafhoppers (). 1970; Pollard, 1970; Saxena and Chada, 1971). Mittler (1957) The mandibular stylets of D. citri house the maxillary stylets, observed that the food and salivary canals in aphids merge to which in their turn form the food and salivary canals. The form a common duct near the tip of the maxillary stylets. However, mandibles show little morphological variation with no alterations in the glassy-winged sharpshooter, Homalodisca coagulata (Say) internally in the concave region, but on the external convex region (Hemiptera: Cicadellidae), the common duct was not observed and 10 grooves or teeth can be seen on the distal extremity of the the food and salivary canals were extended until the distal mandibles (Fig. 2B). Similar to Pollard (1970) in his study of P. mali, extremity of the maxillary stylet (Leopold et al., 2003). we did not find these grooves on the maxillary stylets of D. citri. The junction of the two maxillae is crucial for forming the food Ulman and McLean (1986) also observed the same number of and salivary canals (Forbes, 1969; Forbes and Mullick, 1970). This teeth on the mandibles of the pear psyllid, P. pyricola, whereas junction is possible due to the mandibles which house the maxillae, Mora et al. (2001) found them frequently on the mandibles of the and principally to the grooves along the sides of the maxillary planthopper Tagosodes orizicolus (Muir) (Hemiptera: Delphaci- stylets, forming a sinusoidal clasping device (Forbes, 1969; Pollard, dae). In studies on three species of leafhoppers, Tavella and 1973; Tjallingii, 1978; Tavella and Arzone, 1993), also seen in D. citri Arzone (1993) described 9-10 mandibular teeth for Zyginidia (Figs. 2D and 3D). The food and salivary canals of D. citri (FdC and pullula (Boheman), 10e11 for Empoasca vitis Goethe and 20 for SC, respectively) are located in the centre of the maxilla between Graphocephala fennabi Young. In all these studies, the authors the grooves (Go) and along the stylets until fusion occurs to form suggested that these structures may possibly be specialized for the common duct. In aphids the food canal is equally formed by the

Fig. 3. Serial cross-sections of the labium of Diaphorina citri, from the distal end to the proximal portion of the rostrum, are observed by transmission electron microscopy (TEM) (AeH). (A) Opening of the distal extremity of the labium with mandibular stylets in the middle; (B) Pair of mandibular stylets and neural channels; (C) Pair of mandibles and one of the maxillary stylets, beginning of the food canal or common duct; (D) Pair of mandibles and beginning of second maxilla; (E) Beginning of the juxtaposition between the maxillae forming the food and salivary canals; (F) Juxtaposition of the maxillae and formation of the food and salivary canals; (G) Complete separation between the food and salivary canals; (H) cross section of the stylets closest to the base labium. Common duct (CD); food canal (FdC); beginning of food canal (FdC1); groove (Go); mandibular stylets (Md); maxillary stylets (Mx); neural channels (*); salivary canal (SC); beginning of salivary canal (SC1). Bars represent in A and D: 200 mm, in B: 100 mm, in C: 50 mm and in E: 10 mm. E. Garzo et al. / Arthropod Structure & Development 41 (2012) 79e86 85 two maxillary stylets, whereas the salivary canal is formed mainly Backus, A., McLean, D.L., 1982. The sensory systems and feeding behavior of leaf- by one of them (Pollard, 1973). This dissimilarity between the hoppers. I. The aster leafhopper, Macrosteles fascifrons Stal (Homoptera: Cica- dellidae). J. Morph 172, 361e379. maxillae was observed in M. persicae by Forbes (1969) and it can Backus, E.A., McLean, D.L., 1983. The sensory systems and feeding behavior of also be seen in D. citri (Fig. 3E). leafhoppers. II. A comparison of the sensillar morphologies of several species e The average diameter of the food and salivary canals in aphids (Homoptera: Cicadellidae). J. Morph 176, 3 14. Bonani, J.P., Fereres, A., Garzo, E., Miranda, M.P., Appezzato-Da-Gloria, B., were determined by Auclair (1963, 1964) and Forbes and Lopes, J.R.S., 2010. Characterization of electrical penetration graphs of the Asian MacCarthy (1969), varying from 1 to 2 mm for the food canal and citrus psyllid, Diaphorina citri, in sweet orange seedlings. Entomol. Exp. Appl. 0.2e0.4 mm for the salivary canal. Forbes (1969) and Forbes and 134, 35e49. Bové, J.M., 2006. Huanglongbing: a destructive, newly-emerging, century-old Mullick (1970) observed a food/salivary canals ratio of around 2:1 disease of citrus. J. Plant Pathol. 88, 7e37. for M. persicae and the balsam woolly aphid, Adelges piceae (Rat- Boyd, D.W., 2003. Digestive enzymes and stylet morphology of Deraeocoris nig- zeburg). In D. citri, we observed a similar ratio between the food ritulus (Uhler) (Hemiptera: Miridae) reflect adaptations for depredatory habits. Ann. Entomol. Soc. Am. 96, 667e671. and salivary canals, whose mean diameters were 0.9 0.04 and Boyd, D.W., Cohen, A.C., Alverson, D.R., 2002. Digestive enzymes and stylet 0.4 0.04 mm, respectively. Similar diameters of food (1.0 mm) and morphology of Deraerocoris nebulosus (Hemiptera: Miridae), a predacious plant salivary (0.4 mm) canals were reported for A. piceae (Forbes and bug. Ann. Entomol. Soc. Am. 95, 395e401. fl Mullick, 1970). However, Forbes (1969) found larger dimensions Bradley, R.H.E., 1962. Response of the aphid Myzus persicae to some uids applied to the mouthparts. Can. Entomol. 94, 707e722. for the same ducts (1.2 and 0.5 mm in diameter, respectively) in Brady, J., 1965. A simple technique for making very fine, durable dissecting needles M. persicae, which are above the average reported for aphids. In by sharpening tungsten wire electrolytically. Stain. Technol. 35, 105e106. Capoor, S.P., Rao, S.P., Viswanath, S.M., 1967. Diaphorina citri Kuway., a vector of the Auchenorrhyncha, these duct diameters are even larger: 2.6 and 1.2 e m m greening disease of citrus in India. Indian J. Agric. Sci. 37, 572 576. in Z. pullula; 2.0 and 1.0 minE. vitis; and 13.0 and 4.0 min Evert, R.F., Eschrich, W., Eichhorn, S.E., Limbach, S.E., 1973. Observations on pene- G. fennabi for the alimentary and salivary canals, respectively tration of barley leaves by the aphid Rhopalosiphum maidis (Fitch). Protoplasma e (Tavella and Arzone, 1993), reaching 6.2 and 20.8 mm for the food 77, 95 110. Feeman, T.P., Buckner, J.S., Nelson, D.R., 2000. Stylet length of whitefly adults and canal of 1st instar nymphs and adults of H. coagulata (Leopold et al., nymphs and the mechanism of stylet insertion into the leaves of host plants. 2003). Microsc. Microanal 6 (suppl2), 876e877. The newly described anatomical structure in the common duct Feeman, T.P., Buckner, J.S., Nelson, D.R., Chu, C.C., Henneberry, T.J., 2001. Stylet penetration by Bemisia argentifolii (Homoptera: Aleyrodidae) into host leaf of the aphid maxillary stylets, called the acrostyle by Uzest et al. tissue. Ann. Entomol. Soc. Am. 94, 761e768. (2010), is unlikely to be present in the stylets of D. citri. Uzest Forbes, A.R., 1969. Stylets of the green peach aphid. Myzus persicae. The Can. et al. (2010) proposed that some non-circulative viruses might Entomol. 101, 31e41. Forbes, A.R., 1972. Innervation of the stylets of the pear psylla, Psylla pyricola use the acrostyle to interact with their aphid vectors to ensure (Homoptera: Psyllidae), and the greenhouse whitefly, Trialeurodes vaporariorum plant-to-plant transmission. However, in D. citri the acrostyle-like (Homoptera: Aleyrodidae). J. Entomol. Soc. Brit 69, 456e488. structure is probably absent as this insect is unable to transmit Forbes, A.R., McCarthy, H.R., 1969. Morphology of the Homoptera, with emphasis on stylet-borne non-circulative viruses. Psyllids are vectors of phloem- virus vector. In: Maramorosch, K. (Ed.), Viruses, vectors, and vegetation. Inter- science, N. Y., p. 666. restricted bacteria such as and liberibacters, which Forbes, A.R., Mullick, D.B., 1970. The stylets of the balsam woolly aphid, Adelges are transmitted in a circulative-propagative manner (Weintraub piceae (Homoptera: Adelgidae). Can. Entomol. 102, 1074e1082. and Beanland, 2006; Gottwald et al. 2007), but have never been Foster, S., Goodman, L.J., Duchett, J.G., 1983. Ultrastructure of sensory receptors on the labium of the rice brown planthopper. Cell Tissue Res. 230, 353e366. reported as vectors of plant viruses. Therefore, the acrostyle-like Gottwald, T., Rda Graça, J.V., Bassanezi, R.B., 2007. Citrus huanglongbing: the structure present in the common duct might be specificto pathogen and its impact. Plant Health Prog.. doi:10.1094/PHP-2007-0906-01-RV aphids, which have developed a specific structure in the cuticle On line. Grove, A.J., 1920. The anatomy of the head and mouthparts of Psylla mali, the apple possibly to interact with non-circulative plant viruses. As a conse- sucker, with some remarks on the function of the labium. Parasitology 11, quence, this study revealed important information about the 456e488. mouthparts of D. citri, which will contribute to a better under- Halbert, S.E., Manjunath, K.L., 2004. Asian citrus psyllids (Sternorrhyncha: Psyllidae) and greening disease of citrus: a literature review and assessment of risk in standing of the insect vector-pathogen-host interactions and serve Florida. Flor. Entomol. 87, 330e353. as a basis for future research. Leopold, R.A., Freemanb, T.P., Bucknera, J.S., Nelsona, D.N., 2003. Mouthpart morphology and stylet penetration of host plants by the glassy winged sharpshooter, Homalodisca coagulata, (Homoptera: Cicadellidae). Arthropod Acknowledgements Struct. Dev. 32, 189e199. Maranhão, Z.C. (Ed.), 1978. Morfologia geral dos insetos. Livraria Nobel S. A., São Paulo, p. 396. The authors thank F. Pinto, C. Morcillo and the Electron Mittler, T.E., 1957. 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