Arthropod Structure & Development 41 (2012) 79e86 Contents lists available at ScienceDirect Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd Morphological description of the mouthparts of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: 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 Diaphorina citri, 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. Liberibacter 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 aphid 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 aphids (Hemiptera: Aphididae) 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 insect 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 À210 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 À90 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 leafhoppers sample was imaged at 15 kV in the precooled chamber at À150 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 insects 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 planthoppers (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 leafhopper 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 Cacopsylla. 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 psylla, 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).