Digestive, Salivary, and Reproductive Organs of Bemisiatabaci (Gennadius) (Hemiptera: Aleyrodidae) B TypeMurad Ghanim,1 Rosemarie C. Rosell,2 Leslie R. Campbell,4 Henryk Czosnek,1 Judith K. Brown,3and Diane E. Ullman4*

1Department of Field Crops and Genetics, The Hebrew University of Jerusalem, Rehovot, Israel2Department of Biology, University of St. Thomas, Houston, Texas3Department of Plant Sciences, The University of Arizona, Tucson, Arizona4Department of Entomology, University of California, Davis, California

ABSTRACT A microscopic analysis of the morphologyand ultrastructure of the digestive, salivary, and repro-ductive systems of adult Bemisia tabaci (Gennadius)(Hemiptera: Aleyrodidae) B type was conducted usinglight, scanning, and transmission electron microscopy.The internal anatomy of B. tabaci was found to be similarto that reported for Trialeurodes vaporariorum. In a mi-croscopic analysis of the salivary glands, we have shownthat each primary salivary gland is composed of at least 13cells varying in morphology and staining differentially,while the accessory salivary glands are composed of fourmorphologically similar cells. We analyzed the course ofthe alimentary canal in B. tabaci, demonstrated the inter-nal morphology of the organs, and clarified the location ofthe filter chamber relative to other organs in the whitefly.Our observations confirm that the pair of structures ex-tending from the connecting chamber are caeca that mayaid in fluid movement through the midgut and are not

Malpighian tubules, as previously suggested. We confirman earlier finding that the whitefly lacks Malpighian tu-bules, having instead specialized Malpighian-like cellswithin the filter chamber at the juncture with the internalileum. Finally, we provide the first scanning electron mi-croscopic analysis showing the reproductive organs of B.tabaci. Our investigation provides clarified terminologyfor several components of the digestive and excretory sys-tem. We also provide drawings and micrographs that willaid future researchers in localizing the internal organs ofB. tabaci. We expect our analysis to provide a valuable toolfor studying B. tabaci / plant virus interactions and phys-iological and biological aspects of this insect. J. Morphol.248:22–40, 2001. © 2001 Wiley-Liss, Inc.

KEY WORDS: Internal anatomy; silver leaf whitefly;sweet potato whitefly; internal morphology

Members of the Bemisia tabaci (Gennadius) spe-cies complex (Brown et al., 1995; Frohlich et al.,1999) are pests of many crops worldwide (Byrne etal., 1990). These small, but widely distributed in-sects are also a threat to food, fiber, and ornamentalcrops through the transmission of plant viruses(Brown and Bird, 1992; Brown, 1994; Czosnek andLaterrot, 1997). One member of this complex, B.tabaci, B type (Costa and Brown, 1991) or B. argen-tifolii (Perring et al., 1993; Bellows et al., 1994),erupted globally as a pest and vector of begomovi-ruses in the late 1980s and early 1990s (Brown andBird, 1992). The magnitude of crop losses associatedwith this whitefly and its relatively sudden world-wide dissemination awakened a keen global interestin all aspects of its basic biology (Brown and Bird,1992; Gill, 1992; De Barro, 1995; Gilbertson andUllman et al., 1998).

The anatomy and morphology of the feeding struc-tures (Rosell et al., 1995; Hunter et al., 1996) andinternal organs have been of special interest becausedirect plant damage and virus transmission occurduring feeding and viruses are thought to circulatefrom the insect digestive system to the salivary

glands before they may be transmitted (Costa, 1976;Bird and Maramorosch, 1978; Cohen et al., 1983,1989; Yokomi et al., 1990; Brown et al., 1991; Costaand Brown, 1991; Brown, 1996; Rosell et al., 1999).A thorough knowledge of whitefly internal anatomyis a prerequisite to investigating plant virus inter-actions with whitefly cells and organs and to estab-lishing the circulative pathway of viruses in thiswhitefly vector. In addition, information about theanatomy of Bemisia tabaci is essential to determin-ing how this insect functions relative to feeding,nutrition, and plant damage through feeding andplant virus transmission. Ultimately, this knowl-edge may lead to novel ideas about how to reducewhitefly populations and their impact on crops.

Contract grant sponsor: the United States-Israel Binational ScienceFoundation (BSF); Contract grant number: 95-168; Contract grantsponsor: NRI-USDA; Contract grant number: 389110-G-25-511; Con-tract grant sponsor: E. I. Dupont de Nemours Co.

*Correspondence to: D.E. Ullman, Department of Entomology, Uni-versity of California, One Shields Avenue, Davis, CA 95616.E-mail: deullman@ucdavis.edu

JOURNAL OF MORPHOLOGY 248:22–40 (2001)

© 2001 WILEY-LISS, INC.

The aim of this article is to provide a light, scan-ning, and electron microscopic analysis of the inter-nal anatomy of Bemisia tabaci type B that focuses onthe digestive system, reproductive system, and sal-ivary glands. To our knowledge, this study of white-fly internal anatomy is the first to combine light andscanning electron micrograph illustrations of dis-sected organs with light and transmission electronmicroscopic investigation. In addition, the ultra-structure of the primary and accessory salivaryglands are shown for the first time. In essence, weprovide a map of the internal organs of this insectthat will assist in localization of plant viruses in thisvector insect and provide a basis for evaluating thephysiological impact of potential control measuresthat target digestive physiology or components ofthe reproductive system. Finally, we clarify the lo-cations of certain organs relative to earlier reports(Harris et al., 1995, 1996; Cicero et al., 1995), pro-vide new insights into their internal morphologyand possible function and provide a unifying termi-nology that can be used by whitefly researchers.

MATERIALS AND METHODSMaintenance of Whiteflies and Host Plants

Bemisia tabaci type B (Gennadius) (also B. argen-tifolii [Perring et al., 1993]) whiteflies were used forthese investigations. Species were identified at eachlocation by one or more of the following methods:morphological analyses (Rosell et al., 1997), esterasepatterns (Costa and Brown, 1991), or analysis ofmitochondrial markers (Frohlich et al., 1999). Fro-zen vouchers of Israeli and Arizona populations aremaintained by J. Brown, Department of Plant Sci-ences, The University of Arizona, Tucson, Arizona.Voucher specimens of California populations are atthe Bohart Museum, University of California, Davis,California. Whiteflies were reared in laboratory col-onies on cotton, Gossypium hirsutum (L.) in the De-partment of Plant Sciences, The University of Ari-zona and in the Department of Field Crops andGenetics, The Hebrew University of Jerusalem. Inthe Department of Entomology at University of Cal-ifornia at Davis whiteflies were reared on poinset-tias. Whitefly colonies were maintained in a growthroom at ambient temperature under a 16-h photo-period. In Arizona, individual adult whiteflies werecollected from the colony using hand-held aspiratorsand allowed to feed for 48 h (28°C, 16/8 h L:D) onhealthy pumpkin, Cucurbita maxima (Duchesne),plants at the 3–4 leaf stage prior to preparation formicroscopy. In California and Israel, insects wereremoved directly from the colony and prepared formicroscopy observations. Individual adult whiteflieswere collected from the colony using hand-held as-pirators, transferred to microfuge tubes, and heldlive until preparation for microscopy.

Light Microscopy (LM)

Adult whiteflies were prepared for viewing withthe compound microscope using methods of fixationand embedding as described by Westcot et al. (1993)except that specimens were fixed in 4% glutaralde-hyde in phosphate buffered saline (PBS) (pH 7.4).Serial thick sections of 1–2 mm were made usingglass knives on a Sorvall Porter-Blum Ultramic-rotome MT-2, stained with toluidine blue or basicfuchsin and viewed with a Nikon Micro-phot SAcompound microscope.

To better localize and understand the specific con-nections between organs, dissections of adult white-flies were done using the following methods. White-flies were collected by aspiration from plants andtreated with a slight stream of carbon dioxide toarrest movement. Dissections were performed in in-sect physiological saline (IPS) with 1% toluidineblue on a glass slide as described in Bandla et al.(1998) using a Nikon SMZ-U dissection microscope.To observe the digestive and reproductive systems,the insect was pulled apart at the juncture betweenthe prothorax and metathorax using forceps. Thecontents of the abdomen were then expelled from thebody by pushing with the forceps on the abdomentip. To observe the salivary glands, the head of theinsect was pulled away from the prothorax. Organsof interest were then teased away from the body andallowed to absorb the toluidine blue from the solu-tion. The IPS with 1% toluidine was gently removedfrom successful dissections and 0.2% glutaraldehydeand 4% paraformaldehyde were immediately intro-duced to fix the tissues. The dissection was thencovered with a 22 mm sq. No. 1 cover glass andobserved with the compound microscope.

Scanning Electron Microscopy (SEM)

Dissections were performed as described for lightmicroscopy except that IPS was used without tolu-idine blue. Dissected organs were flushed with ster-ile distilled water, fixed with 0.2% glutaraldehydeand 4% paraformaldehyde in PBS for 5 min, dehy-drated with an ascending ethanol series, and treatedwith hexamethyldisilazane (HMDS) (Sigma, Reho-vot, Israel) twice for 45 min each and air-dried over-night. Samples were then gold coated and observedin a JEOL 5410 scanning electron microscope.

Transmission Electron Microscopy (TEM)

Adult whiteflies were collected by aspiration fromplants and immediately cooled to 20°C or treatedwith a slight stream of carbon dioxide to arrestmovement. Whiteflies were loosely attached to aglass slide using clear nail polish and the organswere dissected out under fixative, either 0.5% glu-taraldehyde : 2% paraformaldehyde in 0.05 M so-dium cacodylate buffer (pH 7.4) or 4% glutaralde-hyde in the same buffer. Due to the small size of the

23MICROSCOPIC ANALYSIS OF BEMISIA TABACI ORGANS

tissues, warm 2% Noble Agar (Difco, Detroit, MI)was pipetted over the dissected tissues and allowedto harden. The agar containing the tissues were cutinto blocks and immersed in the appropriate fixa-tive. The tissues were processed by two methods: 1)standard processing for TEM including overnightglutaraldehyde fixation, sodium cacodylate rinses,osmification, buffer rinses, dehydration in an as-cending ethanol series, followed by propylene oxideand embedding in Spurr’s resin (Spurr, 1969); and 2)light fixation in glutaraldehyde:paraformaldehydefor 1–2 h, buffer rinses, dehydration in an ascendingethanol series, and embedding in LR White (TedPella, Redding, CA). Thin sections (60–90 nm) werestained with aqueous uranyl acetate and lead citrate(Reynolds, 1963), and examined in a Philips EM 420electron microscope (Eindhoven, The Netherlands).

RESULTSForegut and Salivary System

Several LM of the whitefly thorax are shown fororientation in Figure 1. In a dorsoventral section ofthe thorax (Fig. 1A), the paired primary salivaryglands can be seen lying on either side of the sube-sophageal ganglion anterior to the thoracic-abdominal ganglionic mass in the prothorax. Thisarea is at the commissure between the subesopha-geal ganglion and thoracic-abdominal ganglionicmass (Fig. 1B). These latter structures are large andcharacteristic and provided good landmarks. Whenviewing thick sections in a dorsoventral orientation,the primary salivary glands appeared in the protho-rax. They were usually visible in several sectionsbecause of their relatively large size. Figure 2 illus-trates the orientation of the primary salivary glandto the subesophageal ganglion, thoracic-abdominalganglionic mass and the digestive system in a para-sagittal view. The paired accessory glands weremuch smaller in comparison and were visible oneither side and slightly anterior to the primarygland, nearly pressing against the anterior mostportion of the prothorax (data not shown; see Harriset al., 1996, fig. 1). The accessory salivary glandswere extremely difficult to distinguish in thick sec-tions with LM because of their small size and orien-tation to the primary salivary glands. The crumenaand the crumena protractor muscles pass through arelatively large opening between the subesophagealganglion and the thoracic-abdominal mass (the com-missure) (Fig. 1B).

The primary salivary glands are lobed and kidney-shaped (Figs. 3A,B, 4A,B); however, they can bepressed into a circular form in situ. In their positiondorsal to the ventral ganglia of the insect they aretightly pressed against the external esophagus as ittraverses the subesophageal ganglion (Figs. 2, 3B)and continues through the thorax to join the filterchamber (Fig. 3C). Each of the primary salivaryglands has a central lumen (Figs. 3A, 4B, 5) thatempties into a duct, denoted the primary salivary

Fig. 1. LM of Bemisia tabaci showing dorsoventral views ofthe thorax. The mesothorax (MT) was labeled for orientation.A: The paired primary salivary glands (PSG) were visible in theprothorax distal to the thoracic-abdominal ganglionic mass(TAGM). The esophagus is external to the filter chamber as ittraverses the head and thorax (denoted the external esophagus)(EE). Scale bar 5 11.3 mm. B: The crumena (C) and crumenamuscles (CM) pass through the commissure (CO) between thesubesophageal ganglion (SEG) and TAGM. A longitudinal sectionof the external esophagus is present in the metathorax. Scalebar 5 10.2 mm.

24 M. GHANIM ET AL.

gland duct (Figs. 3A, 4B). This duct lies sagittal tothe subesophageal ganglion (Fig. 3A) and curvesunder the ganglion to join the accessory salivarygland duct and the medial duct. We have not de-scribed the connection of the primary salivary ductto the medial, afferent, or efferent ducts or to theaccessory glands because this is well described inHarris et al. (1996).

LM observations of dissected primary salivaryglands (Fig. 4A,B) revealed that the glands are madeof large cells that vary in density, size, and morphol-ogy. These observations were further supported byTEM (Fig. 5). A longitudinal section through one ofthe primary salivary glands revealed a well-definedbasement membrane and at least 13 nearly symmet-rical cells that stained differentially and led to acentral lumen lined with microvilli (Fig. 5). Thislumen empties into a duct at the base of the gland.

The cells are numbered in Figure 5 to facilitatethe following description. Cells 1 and 2 are similar insize and shape and larger than other cells in thegland. They are characterized by small, electron-dense granules and exceptionally large nuclei. Inaddition, the cytoplasm of these cells is less densethan the remaining cells in the gland. Cells 3–11 allhave many small electron-dense granules. Cells 3and 4 are distinguished by a predominance of disor-ganized rough endoplasmic reticulum. In contrast,cells 5–8 have large electron-dense granules, lipid-like accumulations, and whorls of organized roughendoplasmic reticulum. Cell 9 is smaller than manyof the other cells and appears round in this section.It differs from all the other cells in that it has ahigher concentration of lipid-like accumulations,has few organized whorls of rough endoplasmic re-ticulum, and many very fine electron-dense gran-ules. Cells 10 and 11 are extremely small in this

section, but seem to be primarily composed of fine,electron-dense granules. Cells 12 and 13 have a com-paratively dense cytoplasm formed by numeroustightly packed, electron-dense granules and roughendoplasmic reticulum. The dense cytoplasm,heavily laden rough endoplasmic reticulum, and ve-siculation in these cells suggest they may be special-ized secretory cells.

The accessory glands are quite small and simplein structure when compared to the primary glands.Figure 6 shows a cross section through one accessorysalivary gland. Four cells, similar to one another insize, morphology, and content, encircle a central lu-men lined with extensive microvilli. Dark, denselystaining secretions are visible in the lumen of thegland. The cytoplasm of each cell is packed withelectron-dense and electron-lucent vesicles and thecells have large nuclei, although not as large as cellsin the primary salivary gland. The accessory glandcells do not have organized whorls of rough endo-plasmic reticulum nor the dense cytoplasm thatcharacterized some cells in the primary salivaryglands.

Digestive System

For our discussion, the alimentary canal beginswhere the stylet food canal empties into theprecibarium and cibarium. The cibarium and sali-vary syringe are found beneath the subesophagealganglion (Fig. 3B). The cibarium empties into thepharynx. which traverses the circumesophageal pas-sage and the tentorial bar (Figs. 2, 3B), and ulti-mately joins the external esophagus (that portion ofthe esophagus external to the filter chamber) (Fig.3B,C).

Fig. 2. Drawing of a parasagittal view of an adult Bemisia tabaci showing the digestive system in a nongravid individual and thelocation of one primary salivary gland in the prothorax. Note: In gravid females the midgut was sometimes found in the thorax pressedagainst the primary salivary glands. AM, ascending midgut; CA, caeca; CC, connecting chamber; CIB, cibarium; DM, descendingmidgut; EE, external esophagus; FC, filter chamber; HG, hindgut; PSG, primary salivary gland; PSGD, primary salivary gland duct;RS, rectal sac; SEG, subesophageal ganglion; TAGM, thoracic-abdominal ganglionic mass.

25MICROSCOPIC ANALYSIS OF BEMISIA TABACI ORGANS

The external esophagus is extremely slender andtraverses the length of the thorax across the dorsalside of the subesophageal and thoracic-abdominalganglionic masses (Figs. 2, 3B). It is formed by thinepithelial cells with a well-defined basal membraneand a heavy cuticular intima lining surrounding thelumen (Fig. 7). The external esophagus has substan-tial musculature and the cuticular intima lining isgenerally folded accordion-like when these musclesare relaxed (Fig. 7). The esophagus enters the filterchamber anterior to the connecting chamber, atwhich point it is denoted the internal esophagus(Figs. 3C, 8, 9A,C). Figures 2 and 10 show the rela-tionships between these organs in parasagittal andcross section views, respectively.

SEM observations reveal the external aspects ofthe junction between the esophagus, filter chamber,connecting chamber, and caeca (Fig. 8). Portions ofthe descending and ascending midgut and the hind-gut where it is external to the filter chamber are alsoshown (Fig. 8). The internal anatomy of the filterchamber is shown in a TEM montage (Fig. 9) and adrawing (Fig. 10). The filter chamber was formed bya sheath of thin epithelial cells with a thin basalmembrane and what appears to be a cuticular in-tima lining (Fig. 9A). The internal surface of thesecells face the continuous lumen of the filter chamber.A layer of epithelial cells on the opposing side of thecontinuous lumen appears to originate from the as-cending midgut. These cells have extensive mi-

Fig. 3. LM of Bemisia tabaci showing parasagittal (A,B) and dorsoventral (C) views of the head and thorax. A: One primarysalivary gland (PSG) in the prothorax (PT) is visible. The primary salivary gland duct (PSGD) is sagittal to the subesophageal ganglion(SEG). The circumesophageal passage (CEP) between the brain (B) and subesophageal ganglion is visible. Scale bar 5 20 mm. B: Oneprimary salivary gland in close association with the external esophagus (EE) where it traverses the thorax, dorsal to the ventralganglia. The subesophageal ganglion is present in this section. The cibarium (CIB) and salivary syringe (SS) are ventral to thecircumesophageal passage. Scale bar 5 5.15 mm. C: The external esophagus traverses the mesothorax (MT), after which it enters thefilter chamber (FC), expands rapidly, and is denoted the internal esophagus (IE). The location of the filter chamber and othercomponents of the digestive system were variable depending on developmental factors such as ovary and egg development. In thisindividual, a portion of the filter chamber is in the metathorax and extends into the abdomen. The unmarked arrows indicate thejuncture between the metathorax and the abdomen. The epithelial cells forming a sheath (S) around the filter chamber are visible.Arrowhead 1 shows where the internal esophagus begins. Arrowhead 2 indicates where the internal esophagus shares the continuouslumen within the filter chamber with other components of the digestive system. The asterisks show where the connecting chambermeet the filter chamber and the descending midgut (DM) begins. A small section of the ascending midgut (AM) is also present. Scalebar 5 11.2 mm.

26 M. GHANIM ET AL.

crovilli that extend into the continuous lumen (Figs.9A,B, 10). Their basal plasma membrane interdigi-tate with the basal plasma membrane of an innerlayer of epithelial cells in extensive polygonal infold-ings (Figs. 9A–C, 10). The epithelial cells formingthis inner layer surround the lumen of the ileumwhere it is internal to the filter chamber (the inter-nal ileum) (Figs. 9A,B, 10). Some Malpighian-likecells, all with extensive microvilli, adjoin the epithe-lial cells of the internal ileum (Figs. 9A,C, 10). As in

Cicero et al. (1995), the designation Malpighian-likeis used for these cells because they have well-developed microvilli, extensive polygonal infoldings,basal labyrinth, large nuclei, and extensive septatedesmosomes that are common to Malpighian tu-bules. In addition, their location is consistent withan excretory function. Ingested plant materials andcrystallized substances were consistently present inthe continuous lumen (Fig. 9A), but were not ob-served in the central lumen of the internal ileum.

The internal esophagus expands within the filterchamber where it is united with the continuous lu-men (Figs. 9A,C, 10). The continuous lumen appearsto extend into the connecting chamber, caeca, anddescending and ascending midguts (Figs. 10, 11).The ascending midgut then enters the filter cham-ber and meets the continuous lumen and internalileum, as described above.

Within the connecting chamber, the continuouslumen is highly convoluted, extending into the caecaand the descending midgut (Figs. 10, 11A–C). Thedescending midgut exits the connecting chamber(Figs. 10, 11A) and maintains a large diameter to itsbase where it meets the ascending midgut (Fig.11A). The ascending midgut narrows in diameteruntil it enters the filter chamber where it meets boththe continuous lumen and the internal ileum (Figs.10, 11A,C). Hence, the midgut does not loop back onitself, but rather the ascending midgut adjoins theileum within the filter chamber (see above).

Extending from the connecting chamber are a pairof caeca (incorrectly denoted Malpighian tubules byHarris et al., [1995, 1996]) (Figs. 2, 10, 11). Thecaeca have extensive musculature that absorbsstain quickly in dissection (Fig. 11A,B) and a largelabyrinth-like lumen (Fig. 11B) surrounded by ex-tremely thin epithelial cells that have what appearto be a cuticular intima lining (data not shown).

The descending midgut is composed of exception-ally thick epithelial cells surrounding a large lumen(Fig. 12). The epithelial cells have a relatively thinbasement membrane facing the insect hemocoel(Fig. 12) and a vesiculated, pleated apical membranethat resembles disorganized, sparsely placed mi-crovilli extending into the lumen (Fig. 12, inset).There are longitudinal muscles occasionally presenton the descending midgut, but circular muscles werenot observed (Fig. 12). Many vacuoles and vesiclesare present and the cells have exceptionally largenuclei with well-developed nucleoli. Crystalline ma-terials were nearly always observed in the descend-ing midgut lumen (Fig. 12).

The ascending midgut is smaller in diameter thanthe descending midgut and is formed by very thickepithelial cells surrounding a rather small lumen(Fig. 13). In contrast to the descending midgut, theascending midgut has a well-organized and exten-sive brush border of microvilli. In some areas thereare multiple pleated membranes extending beyondthe microvilli. These pleated membranes may beextensions of the microvilli or possibly part of a

Fig. 4. LM of dissected primary salivary glands (PSG) fromBemisia tabaci. A: The primary salivary glands (PSG) are pairedand kidney-shaped when dissected and often remain attached tothe feeding apparatus (stylet bundle [SB], labrum [LBR], andcibarium [CIB]) by their ducts. Scale bar 5 9.7 mm. B: Magnifi-cation of one primary salivary gland reveal large, morphologicallyvariable cells that stain differentially and the primary salivarygland duct (PSGD) as it exits the gland. Scale bar 5 5.2 mm.

27MICROSCOPIC ANALYSIS OF BEMISIA TABACI ORGANS

peritrophic membrane. The nuclei of the ascendingmidgut epithelial cells are large and sometimes havemultiple nucleoli. A strikingly high number ofelectron-lucent vacuoles and vesicles are character-istic of the ascending midgut, as well as smallelectron-lucent vesicles carrying electron-densegranules. Crystalline material similar to that in

other regions of the midgut and continuous lumen ofthe filter chamber are also present in the ascendingmidgut. Like the descending midgut, there are fewlongitudinal muscles lining the ascending midgut.The ascending midgut enters the filter chamber andshares the continuous lumen joining this portion ofthe alimentary canal with the lumen of the internal

Fig. 5. TEM of Bemisia tabaci showing the internal morphology of one primary salivary gland. The gland is composed of at least13 large cells that stain differentially and surround a central lumen (L) lined with microvilli (MV). The gland is nearly symmetricalwith regard to placement of cell types. The cells are numbered for reference to their description in the text. BM, basal membrane; EDG,electron-dense granules; LLA, lipid-like accumulations; N, nucleus; RER, rough endoplasmic reticulum. Scale bar 5 5.4 mm.

28 M. GHANIM ET AL.

Fig. 6. TEM of Bemisia tabaci showing one accessory salivary gland composed of four large cells surrounding a central lumen (L)lined with microvilli (MV). The cells are numbered for reference in the text and are characterized by large nuclei (N) and manyelectron-dense and electron-lucent vesicles. Unmarked arrows indicate dark secretions in the central lumen. Scale bar 5 2 mm.

29MICROSCOPIC ANALYSIS OF BEMISIA TABACI ORGANS

esophagus and the basal membrane of the internalileum (Figs. 9A, 10). Figure 10 shows the internalarrangement of the filter chamber and the proximityof its components to one another. The arrows indi-cate the probable direction in which ingested plantmaterials flow through this system.

The rectal sac has a heavy intima lining (Fig. 14).When relaxed the cells are folded accordion-style.When filled it could expand to a relatively large sizecompared to other portions of the hindgut. The epi-thelial cells are quite thin when the structure isexpanded and have nuclei that are small relative tothe midgut and filter chamber. It has a valve-likestructure with large external muscles that presum-ably contract to open the valve, releasing honeydewvia the rectum and the anus. As is typical of theinsect hindgut, an extensive basal labyrinth is present.

In dissection, the attachment between the filterchamber, connecting chamber, and the descendingand ascending midgut form a loop. This loop ispresent in several locations in the insect, dependingon age and sex. In females prior to extensive eggdevelopment and in males the external esophagusjoins the filter chamber in the metathorax, while thefilter chamber and connecting chamber traverse themetathorax and first abdominal segment (Figs. 2,3C). The descending midgut traverses the abdomen,meeting the ascending midgut in the 7th abdominalsegment. The ascending midgut then returns to thefilter chamber (Fig. 3C). In females with developingeggs, the ovaries, ovarioles, and mature eggs fill theabdomen and crush the hindgut components againstthe exoskeleton on the dorsal surface of the insect.The remaining alimentary components are pushedupward, sometimes entirely into the thorax. Table 1shows the number and age of insects in which thisoccurred in a laboratory experiment in Israel. Thus,

depending on the development and sex of the insect,the entire midgut was found filling the abdomen,folded upon itself in the first few abdominal seg-ments or pushed into the thorax.

Reproductive Organs

The ovaries are formed by a mass of ovarioles (Fig.15A). Each ovariole has series of eggs within whichare in various stages of maturity (Fig. 15A). Asshown in Costa et al. (1996), bacteriocytes enter theeggs via the pedicel end while they are immature(Fig. 15A–C). The egg plasma closes around the bac-teriocyte as it matures. When the egg is fully maturethe bacteriocyte is enclosed in the egg plasma and isvisible at the end of the egg where the pedicel forms(Fig. 15C).

Figure 16 shows the external morphology of thetip of the female abdomen. The anus is beneath thelingula and the ovipositor is composed of two pairs of

Fig. 8. SEM of Bemisia tabaci showing a dorsal view of thefilter chamber (FC) where it joins the connecting chamber (CC).The external esophagus (EE) is slender and expands where itbecomes the internal esophagus (IE) in the filter chamber andjoins the connecting chamber (CC) (not visible externally). Thecaeca (CA) extends into the hemocoel from the connecting cham-ber and the descending midgut (DM) exits the connecting cham-ber and joins the ascending midgut (AM). The ascending midgutjoins the continuous lumen within the filter chamber. The arrowon the hindgut (HG) points toward the anus. Scale bar 5 9.7 mm.

Fig. 7. TEM of Bemisia tabaci showing a cross section of theesophagus where it is external to the filter chamber. The esoph-agus has a cuticular intima lining (IN) and is formed by a thinlayer of epithelial cells (EC) surrounding a lumen (L). The well-defined basement membrane (BM) and relatively large muscles(M) external to the esophagus are visible. Scale bar 5 950 nm.

30 M. GHANIM ET AL.

Figure 9

31MICROSCOPIC ANALYSIS OF BEMISIA TABACI ORGANS

gonapophyses. The external morphology of a dis-sected spermatheca at the SEM level is shown inFigure 17A. The spermatheca is found ventrally inthe tip of the abdomen. When the spermatheca wasdissected the sperm within were readily visible witha compound light microscope (Fig. 17B).

Figure 18 shows the external morphology of themale genitalia. A pair of parameres form the tip ofthe male abdomen and the penis is found betweenthe parameres (Fig. 18A,B). As in the female, theanus can be found beneath the lingula on the dorsalside of the insect (Fig. 18B).

DISCUSSION

Our microscopic analysis confirms the location ofthe salivary glands and shows their ultrastructurefor the first time. We resolve a number of controver-sies regarding the anatomy and morphology of thedigestive system and show the morphology of thereproductive organs. By combining light, scanning,and electron microscopy techniques, we provide im-ages for recognition of Bemisia tabaci internal or-gans in dissection and thick and thin sections. Inaddition, we provide drawings that can be used tohelp localize various components of the digestivetrack, understand the organization of the filter

chamber, and assess the movement of fluids throughthe whitefly digestive system. We expect our analy-sis to provide a useful tool for tracking begomovi-ruses through the whitefly and for isolating anddissecting whitefly internal organs for physiologicalstudies.

The most extensive previous analysis of Aleyrodi-dae internal anatomy can be found in Weber (1935).Although that monograph deals with the anatomy ofTrialeurodes, it serves as an extremely useful refer-ence and model for analyzing the internal anatomyof Bemisia spp. Due to the technical limitations ofthe time, the anatomical features are illustratedsolely with line drawings. Although of exceedinglyhigh quality, interpretation of the line drawings rel-ative to the actual appearance of the organs in thewhitefly is often difficult. Our micrographs used inconjunction with these line drawings should be use-ful to those seeking to identify specific whitefly or-gans.

Foregut and Salivary System

Our light microscopic analysis of the feedingstructures, foregut and salivary glands of Bemisiatabaci confirm earlier descriptions (Rosell et al.,1995; Harris et al., 1995, 1996; Hunter et al., 1996).TEM analysis of the primary and accessory salivaryglands revealed a tremendous variation between thecells making up these organs (Figs. 4–6), a findingthat is consistent with what is known about thesalivary glands of other Hemipterans (Wayadandeet al., 1997). The primary glands of the whitefly arecomposed of at least 13 cells that differ considerablyin shape, size, and contents, a finding that suggestsdiverse functions (Figs. 4, 5). In the primary salivarygland, the dense cytoplasm with vesiculation, largenumber of electron-dense granules, and organizedwhorls of rough endoplasmic reticulum is consistentwith high metabolic activity and secretory proteinsynthesis (Palade, 1975). These morphological char-acteristics suggest that the primary salivary glandsmay be the site where enzymatic products of thesaliva are produced. In contrast, the accessory glandis composed of just four cells, each having a verysimilar morphology and content (Fig. 6). Many of thecharacteristics we observed in the accessory salivaryglands are usually associated with cells engaged inpassive water transport, e.g., large numbers ofelectron-lucent vesicles, few electron-dense vesicles,numerous mitochondria, and infolded basal plasmamembranes. Based on these morphological charac-teristics, we speculate that the accessory salivaryglands have osmoregulatory functions or contributeto the watery portion of the saliva (Miles, 1972).

Although there are no detailed studies of whiteflysaliva, the morphology we describe suggests that theprimary and accessory glands have different func-tions. We speculate that the primary salivary glandsactively produce, store, and deliver diverse enzymesimportant in feeding and that the accessory glands

Fig. 9. TEM montage of Bemisia tabaci showing the internalmorphology of the proximal portion of the filter chamber wherethe internal esophagus (IE), internal ileum (II), and ascendingmidgut (AM) converge. A: The filter chamber is formed by asheath (S) of thin epithelial cells with a thin basement membrane(BM) and a cuticular intima lining (IN). The intima lining facesthe continuous lumen (CL) that unites the internal esophagus(IE) with the connecting chamber (not visible) and the midgut.The opposing side of the continuous lumen is formed by a layer ofepithelial cells that appear to originate from the ascending mid-gut (AM). These cells have extensive microvilli (MV) that extendinto the continuous lumen. Their basal plasma membrane inter-digitates the basal plasma membrane of a second layer of cellsthat appear to originate from the internal ileum. The internalileum cells has extensive microvilli (MV) that extend into itslumen (IIL). Malpighian-like cells (MLC) border the internal il-eum. The external esophagus (EE) is present in cross section. Twofood boli (FB) are present in the continuous lumen, but none arefound in the internal ileum lumen. Scale bar 5 4.5 mm. The areasin boxes B and C are enlarged in 9B and C, respectively. B: Thejunction between the two epithelial layers that arise from theascending midgut and the internal ileum (II). Microvilli reminis-cent of a brush border are present on the apical surface of theascending midgut, which border the continuous lumen. The mi-crovilli at the apical surface of the internal ileum epithelium,which border the internal ileum lumen, are less well-organizedthan those associated with the ascending midgut cells. The un-marked arrowheads indicate where the basal plasma membranesof both epithelial layers are highly infolded. Scale bar 5 1.9 mm.C: The convoluted lumen of the internal esophagus where itmeets a Malpighian-like cell. Unmarked arrowheads indicateextensive polygonal infoldings between the basal plasma mem-branes of the Malpighian-like cell and the internal ileum epithe-lia. Cells have large nuclei (N) and disorganized microvillipresent on the apical plasma membrane. Septate junctions (SD)are abundant on the lateral portions of the cell. Scale bar 5 1.9mm. M, muscle.

32 M. GHANIM ET AL.

absorb and deliver substances made elsewhere inthe insect’s body. Along this line of reasoning, theenvironment in the accessory salivary glands maybe ideal to absorb virions from the hemolymph, com-partmentalize these in vesicles, and deliver themwith the watery secretions emanating from thisgland. These are speculations, but they are consis-tent with our observations and with findings inother vector/virus systems, e.g., the aphid/luteovirussystem (Garret et al., 1996; Gildow, 1982, 1993).

Digestive System

Diverse and sometimes contradictory terms havebeen used to denote various parts of the whiteflydigestive system (Cicero et al., 1995; Harris et al.,1995, 1996). We specifically clarify the location ofthe filter chamber and its proximity and connectionsto other parts of the digestive system (Figs. 2, 10).Furthermore, we confirm the finding of Cicero et al.(1995) suggesting the presence of Malpighian-like

Fig. 10. Drawing of a cross section through the filter chamber (FC) of Bemisia tabaci illustrating the components of the digestivesystem present in this region and the probable flow of ingested plant materials. Hatched arrowheads indicate how ingested plantmaterials empty from the external esophagus (EE) into the continuous lumen (CL) of the filter chamber via the internal esophagus(IE). The curved arrows indicate that the majority of the entering plant materials move from the continuous lumen of the filterchamber into the lumen of the adjoining connecting chamber (CC). Ingested plant materials then enter the lumen of the caeca (CA)(curved arrows). The caeca are blind-ended and have been observed to pump during ingestion; thus, the ingested plant materials areforced to circulate back into the connecting chamber lumen and then to enter the descending midgut (DM) (hatched arrowhead). Thesolid black arrowhead indicates movement of plant materials back to the filter chamber via the ascending midgut (AM). The dottedlines with arrowheads at each end indicate that ingested fluids, before or after circulation, may also be absorbed by the ascendingmidgut cells. The basal membrane of these cells interdigitates the basal membrane of the internal ileum (II) cells (double asterisk).Fluids that enter these cells are presumably filtered across the internal ileum and exit the filter chamber via the internal ileum lumen(IIL) which enters the lumen of the hindgut (HG) (solid black arrowheads). Although difficult to depict here, the internal ileum formsa tube within the filter chamber that connects to the hindgut, where it exits the filter chamber. Hence, once fluids enter its lumen theydo not reenter the continuous lumen of the filter chamber. MLC Malphigian-like cells, MV microvilli.

33MICROSCOPIC ANALYSIS OF BEMISIA TABACI ORGANS

cells inside the filter chamber instead of Malpighiantubules.

We confirm the conclusions of Cicero et al. (1995)that the two large structures extending from theconnecting chamber are caeca and not Malpighiantubules, as suggested by Harris et al. (1995, 1996)

and Weber (1935). As in Cicero et al. (1995), weobserved Malpighian-like cells within the filterchamber where the internal ileum meets the ascend-ing midgut (Fig. 9A,C). This finding is consistentwith observations of the internal anatomy of manyHemipterans that have filter chambers (Goodchild,1966; Ammar, 1985).

We are in agreement with Cicero et al. (1995)that the caeca are blind-ended extensions from thealimentary canal. The term diverticula is alsoused for similar structures in other piercing-sucking insects with filter chambers, e.g., fulgo-rids (Goodchild, 1966). These structures arethought to be critical to movement of fluids and inassisting ecdysis in several delphacids (Ammar,1985). They may play a similar role in the white-fly. Salvucci et al. (1998), using fluorescent-labeled amino acids and proteins, observed rapidmovement of labeled molecules through the caecaand entire digestive system. In a preliminary ex-periment using various fluorescently labeled solu-tions, Ghanim and Czosnek (unpublished data)and Rosell and Davidson (unpublished data) ob-served fluids moving rapidly into the caeca fromthe connecting chamber. In addition, continuouscontractions of the caeca were observed that re-sulted in the movement of fluorescent label fromthe caeca into the connecting chamber and subse-quently into the descending midgut, through theascending midgut, and ultimately into the hind-gut. Based on these observations we speculatethat fluids flow through the whitefly digestivetract as indicated in Figure 10.

Cicero et al. (1995) discussed a structure adja-cent to the filter chamber denoted the “convolu-tion.” We assume the authors chose this terminol-ogy because of the complex and “convoluted”lumen of this structure. We cannot be certain, butthe interpretation provided in Cicero et al. (1995)suggests that the structure he called the convolu-tion is the structure we denote as the connecting

Fig. 11. LM of Bemisia tabaci showing a ventral view of por-tions of the alimentary tract after dissection. A: The caeca (CA)and descending midgut (DM) extend from the connecting cham-ber (CC). The lumen (L) of the connecting chamber is convoluted(marked with asterisks) and connects the caeca and the descend-ing midgut. The filter chamber is not visible because it wasunderneath the connecting chamber in this dissection. The as-cending midgut (AM) passes underneath the connecting chamberto join the filter chamber (unmarked arrow). The hindgut (HG)snapped free of the anus during dissection and was consequentlyfolded accordion-fashion underneath the connecting chamber.Scale bar 5 4.9 mm. B: Higher magnification of one caecum. Theassociated muscles (M) stained densely and contrasted with thelabyrinth-like lumen (LA) of the caecum. Asterisks indicate aportion of the lumen. Scale bar 5 4.8 mm. C: Higher magnificationof the ventral face of the connecting chamber (CC) reveal thelumen where it intersects the caeca and descending midgut. Theunmarked arrow shows where the ascending midgut passes un-der the connecting chamber to its juncture with the filter cham-ber. Also shown is the hindgut (HG) as it exits the ventral surfaceof the connecting chamber. Scale bar 5 9.9 mm.

34 M. GHANIM ET AL.

Fig. 12. TEM montage showing a cross section of the descending midgut of Bemisia tabaci. The descending midgut is larger indiameter than the ascending midgut (cf. Fig. 13) and is composed of thick epithelial cells with an especially thin basement membraneand a pleated, vesiculated apical membrane that we speculate to be poorly developed microvilli (MV). Numerous electron-lucentvesicles (ELV), mitochondria, and exceptionally large nuclei (N) are present. Crystalline material (unmarked arrows) is present in thelumen (L) and cross sections of longitudinal muscles (M) are visible on the basal surface. Septate junctions (SD) are present betweenepithelial cells. Scale bar 5 520 nm. Inset: Higher magnification of the apical membrane showing what appears to be poorly developedmicrovilli extending into the lumen. Scale bar 5 200 nm.

35MICROSCOPIC ANALYSIS OF BEMISIA TABACI ORGANS

chamber (Figs. 10, 11A,C). We propose the desig-nation “connecting chamber” because it is moredescriptive of the location and more likely functionof the chamber, which is to connect the filterchamber to the midgut and ultimately to the hind-gut. Harris et al. (1995, 1996) considered thestructure we designate as the connection chamberto be part of the midgut, a finding with which weconcur. However, the line drawings by these authorsincorrectly suggest that the hindgut arises from thisstructure directly between the two caeca (see Harris etal., 1996, fig. 13, parts 16, 17, 19). Our observationsdiffer from Cicero et al. (1995) in that we did notobserve a “gateway” from the filter chamber.

Our designations for the components of the di-gestive system are largely in agreement with Ci-cero et al. (1995). Harris et al. (1995, 1996) labelthe regions of the midgut in an opposite orienta-tion. We label the descending midgut as that por-tion exiting the connecting chamber and the as-cending midgut as that portion ascending to thefilter chamber. The midgut may be twisted orpushed into the thorax in gravid females (Table 1).Hence, it is not possible to reliably distinguishbetween the descending and ascending midgut insectioned specimens, unless the connections to thefilter chamber and connecting chamber can be an-alyzed.

Fig. 13. TEM montage of a cross section of the ascending midgut of Bemisia tabaci. The ascending midgut is smaller in diameterthan the descending midgut (cf. Fig. 12) and is composed of thick epithelial cells with exceptionally extensive microvilli (MV) at theapical membrane that extend into a relatively small lumen (L). These microvilli are extensively folded, sometimes forming membra-nous pleats (MP). Septate junctions (SD) are present between epithelial cells. The cells have large nuclei (N) with well-developednucleoli, electron-lucent vesicles (ELV), and extensive vacuoles and vesicles. Many crystalline rods are present in the lumen (L).Longitudinal muscles (M) are occasionally present and the basement membrane is extremely thin. Scale bar 5 2.1 mm.

36 M. GHANIM ET AL.

Our micrographs and drawings (Figs. 2, 8 –11)show the relationship between the filter chamberand the esophagus, connecting chamber, midgut,and hindgut with more detail and clarity than

previously shown. The internal morphology we ob-served is similar to that shown in line drawings byCicero et al. (1995). Our findings differ from Har-ris et al. (1995, 1996) in that the filter chamber

Fig. 14. TEM montage showing the ultrastructure of the rectal sac of Bemisia tabaci. This area of the rectum is formed by adiversity of cells typical of transitional epithelium, with relatively small nuclei (N). The cells have extensive membranous labyrinthsand basal labyrinths (BL). A muscle (M) leads to a valve-like opening (V) from the lumen (L). The lumen has a cuticular intima lining(IN) that is especially heavy in some regions. Scale bar 5 5.3 mm.

37MICROSCOPIC ANALYSIS OF BEMISIA TABACI ORGANS

was always observed joined to the connectingchamber via the continuos lumen. The filter cham-ber and connecting chamber can easily be mis-taken as one organ at the light microscopy level(see Fig. 11). In our investigations, SEM and TEMobservations were needed to clarify the relation-ships between the filter chamber, the connectingchamber, and other portions of the alimentarycanal.

Begomoviruseses have been shown to accumulatein the filter chamber (Hunter et al., 1998) and are

present in the hemolymph, saliva, and honeydew ofBemisia tabaci (Rosell et al., 1999). The aphid/luteovirus model suggests that plant viruses oftenenter the organs of their vectors via receptor medi-ated endocytosis across cell membranes (Gildow,1982, 1993). If a similar mechanism occurs in thewhitefly/begomovirus system, it seems likely thatthe filter chamber, anterior portion of the descend-ing midgut, or the internal ileum are the most likelyregions of the digestive tract for virus uptake tooccur. The esophagus, caeca, continuous lumenwithin the filter chamber, and rectum are lined witha cuticular intima, making them unlikely sites ofvirus uptake. In contrast, the midgut (within andoutside the filter chamber) and internal ileum epi-thelia have a brush border at the apical membranethat would be ideal for viral endocytosis. There arealso times during development, when the salivaryglands are in close proximity to the filter chamberand the midgut, that may be worthy of attentionwith regard to virus passage.

Reproductive Organs

Our microscopic observations of the reproductivesystems of both female and male Bemisia tabacisupport the information in the line drawings of We-ber (1935). We provide the first light and electronmicroscopic images of these organs and have usedthe terminologies proposed by Weber (1935). Asshown in Costa et al. (1996), we also observed whatappear to be bacteriocytes entering the developingoocytes (Fig. 15). This process is of particular inter-est because one begomovirus (tomato yellow leafcurl) has been shown to be transovarially transmit-ted (Ghanim et al., 1998) and interactions betweenthe endosymbiont and transmission of luteovirusesand certain begomoviruses have been indicated (vanden Heuval et al., 1994, 1997; Morin et al., 1999).

TABLE 1. Location of the midgut loop in adult Bemisia tabaciof different ages

Numbersof insectsexamined

Age of adultfemales

Midgut presentin thorax

Midgut presentin abdomen

30 0–1 day 0 3030 3 days 1 2930 5 days 2 2830 7 days 4 2630 10 days 3 2730 15 days 3 27

Fig. 15. LM of dissected ovaries with immature eggs (IME)and mature eggs (ME) within the ovarioles. A: Mature eggsnearly always contain what appears to be a bacteriocyte (BA).The bacteriocytes enter the immature eggs in the region wherethe pedicel is ultimately formed. The paired caeca (CA) of thedigestive system with their labyrinth-like lumen (LA) appear inthe background. Scale bar 5 10.7 mm. B: Higher magnification ofa bacteriocyte entering an immature egg. The caeca with theirlabyrinth-like lumen appear in the background. Scale bar 5 10.7mm. C: Higher magnification of a mature egg with a bacteriocyteinside and a fully formed pedicel (P). Scale bar 5 10.2 mm.

Fig. 16. SEM of a dorsal view of the tip of the abdomen of afemale Bemisia tabaci. The anus is found beneath the lingula(LI). The ovipositor is formed with paired gonapophysis (GO) andvalvifer (ventral to the GO). Scale bar 5 57 mm.

38 M. GHANIM ET AL.

ACKNOWLEDGMENTS

Figures and drawings were prepared by Diane E.Ullman and Rosemarie C. Rosell with assistancefrom Eileen J. Rendahl (University of California),whom we thank for her patient help. We thank MarvKinsey (University of California at Davis) and DavidBentley (University of Arizona) for technical assis-tance in preparation of tissues and interpretation ofdata. We also thank Michael Parrella and David vonDamm-Kattari (University of California) and RafaelCaballero and Ali M. Idris (University of Arizona)for providing whiteflies. This research was sup-ported by grant 95-168 from the United States-Israel Binational Science Foundation (BSF) (toCzosnek and Ullman) and NRI-USDA under award#389110-G-25-511 (to Brown and Rosell). Initial

studies toward this project were also generouslysupported through a grant from E. I. Dupont deNemours Company, Newark, DE.

LITERATURE CITED

Ammar ED. 1985. Internal morphology and ultrastructure ofleafhoppers and planthoppers. The leafhoppers and planthop-pers. New York: Wiley-Interscience. p 127–162.

Bandla MD, Campbell LR, Ullman DE, Sherwood JL. 1998. In-teraction between tomato spotted wilt virus Tospovirus (TSWV)glycoproteins with a thrips midgut protein: a potential cellularreceptor for (TSWV). Phytopathology 88:98–104.

Bellows TS, Thomas JR, Perring M, Gill RJ, Headrick DH. 1994.Description of a species of Bemisia (Homoptera: Aleyrodidae).Ann Entomol Soc Am 87:195–206.

Bird J, Maramorosch K. 1978. Viruses and virus diseases associ-ated with whiteflies. Adv Virus Res 22:55–110.

Brown JK. 1994. The status of Bemisia tabaci (Genn.) as a pestand vector in world agroecosystems. FAO Plant Prot Bull 42:3–32.

Brown JK. 1996. Molecular biology and epidemiology of SubgroupIII, Geminiviridae. In: Stacey G, Keen N, editors. Plant-microbeinteractions review series. London: Chapman and Hall. p 125–195.

Brown JK, Bird J. 1992. Whitefly-transmitted geminiviruses andassociated disorders in the Americas and the Caribbean Basin.Plant Dis 76:220–225.

Fig. 17. SEM and LM of the spermatheca of Bemisia tabaci.This structure is found ventrally in the tip of the abdomen.A: SEM of the external surface of the spermatheca. Scale bar 514.2 mm. B: LM of dissected spermatheca with sperm (SP). Scalebar 5 1.98 mm.

Fig. 18. SEM of the tip of the abdomen of a male Bemisiatabaci. A: Sagittal view of the parameres (PA) and the penis (PE).Scale bar 5 20 mm. B: Dorsal view of the parameres (PA) and thepenis (PE). The anus is beneath the lingula (LI). Scale bar 5 47.5mm.

39MICROSCOPIC ANALYSIS OF BEMISIA TABACI ORGANS

Brown JK, Costa HS, Laemmlen F. 1991. First incidence ofwhitefly-associated squash silverleaf (SSL) of Cucurbita, and ofwhite streaking (WSt) disorder of cole crops in Arizona andCalifornia. Plant Dis 76:426.

Brown JK, Frohlich DR, Rosell RC. 1995. The sweetpotato orsilverleaf whiteflies: biotypes of Bemisia tabaci or a specialcomplex. Annu Rev Entomol 40:511–534.

Byrne DN, Bellows TS Jr, Parrella MP. 1990. Whitefly in agri-cultural systems. In: Gerling D. Whiteflies: their bionomics,pest status and management. Andover, UK: Intercept. p 227–261.

Cicero JM, Hiebert E, Webb SE. 1995. The alimentary canal ofBemesia tabaci and Trialeurodes abutilonea (Homoptera, Ster-norrhynchi): histology, ultrastructure and correlation to func-tion. Zoomorphology 115:31–39.

Cohen S, Duffus JE, Larsen RC, Liu HY, Flock RA. 1983. Puri-fication, serology, and vector relationships of squash leaf curlvirus, a whitefly-transmitted geminivirus. Phytopathology 73:1669–1673.

Cohen S, Duffus JE, Liu HY. 1989. Acquisition, interference, andretention of cucurbit leaf curl viruses in whiteflies. Phytopa-thology 79:109–113.

Costa AS. 1976. Whitefly-transmitted plant diseases. Annu RevPhytopathol 14:429–449.

Costa HS, Brown JK. 1991. Variation in biological characteristicsand esterase patterns among populations of Bemisia tabaci,and the association of one population with silver leaf symptominduction. Ent Exp Appl 61:211–219.

Costa HS, Toscano NC, Henneberry TJ. 1996. Mycetocyte inclu-sion in the oocytes of Bemisia argentifolii (Homoptera: Aleyro-didae). Ann Entomol Soc Am 89:694–699.

Czosnek H, Laterrot H. 1997. A worldwide survey of tomatoyellow leaf curl viruses. Arch Virol 142:1391–1406.

De Barro PJ. 1995. Bemisia tabaci biotype B: a review of itsbiology, distribution, and control. Second Edition Common-wealth Scientific and Industrial Organisation Division of Ento-mology: Technical Paper No 36:1–58.

Frohlich DI, Torres-Jerez ID, Bedford PG, Markham, Brown JK.1999. A phylogeographic analysis of the Bemisia tabaci speciescomplex based on mitochondrial DNA markers. Molec Ecol8:1593–1602.

Garret A, Kerlan C, Thomas D. 1996. Ultrastructure study ofacquisition and retention of potato leafroll luteovirus in thealimentary canal of its aphid vector, Myzus persicae Sulz. ArchVirol 141:1279–1292.

Ghanim M, Morin S, Zeidan M, Czosnek H. 1998. Evidence fortransovarial transmission of tomato yellow leaf curl virus by itsvector the whitefly Bemisia tabaci. Virology 240:295–303.

Gilbertson RL, Ullman DE, Salati R, Maxwell DP, Grafton-Cardwell EE, Polek M. 1998. Insect transmitted virusesthreaten agriculture. Calif Agri 52:23–28.

Gildow F. 1982. Coated-vesicle transport of luteoviruses throughsalivary glands of Myzus persicae. Phytopathology 72:1289–1296.

Gildow F. 1993. Evidence for receptor-mediated endocytosis reg-ulating luteovirus acquisition by aphids. Phytopathology 83:270–277.

Gill R. 1992. A review of the sweetpotato whitefly in southernCalifornia. Pan-Pac Entomol 68:144–152.

Goodchild AJP. 1966. Evolution of the alimentary canal in theHemiptera. Biol Rev 41:97–140.

Harris KF, Pesic-van esbroeck Z, Duffus JE. 1995. Anatomy of avirus vector. Bemisia 1995: taxonomy, biology, damage controland management. London: Intercept. p 289–318.

Harris KF, Pesic-van esbroeck Z, Duffus JE. 1996. Morphology ofthe whitefly Bemisia tabaci (Homoptera: Aleyrodidae) relativeto virus transmission. Zoomorphology 116:143–156.

Hunter W, Hiebert E, Webb SE, Polton JE, Tsai HT. 1996.Precibarial and cibarial chemosensilla in the whitefly, Bemisiatabaci (Gennadius) (Homoptera: Aleyrodidae). Int J Insect Mo-phol Embryol 25:295–304.

Hunter WB, Hiebert E, Webb SE, Tsai JH. 1998. Location ofgemniviruses in the whitefly, Bemisia tabaci (Homoptera: Aley-rodidae). Plant Dis 82:1147–1151.

Miles AS. 1972. The saliva of Hemiptera. Adv Insect Physiol9:183–255.

Morin S, Ghanim M, Zeidan M, Czosnek H, Verbeek M, van denHeuvel JFJM. 1999. A GroEL homologue from endosymbioticbacteria of the whitefly Bemisia tabaci is implicated in thecirculative transmission of tomato yellow leaf curl virus. Virol-ogy 30:75–84.

Palade G. 1975. Intracellular aspects of the process of proteinsecretion. Science 189:347–358.

Perring TM, Cooper AD, Rodriguez RJ, Farrar CA, Bellows TS.1993. Identification of a whitefly species by genomic and behav-ioral studies. Science 259:74–77.

Reynolds ES. 1963. The use of lead citrate at high pH as anelectron opaque stain in electron microscopy. J Cell Biol 43:664–671.

Rosell RC, Lichty JE, Brown JK. 1995. Ultrastructure of themouthparts of adult sweetpotato whitefly, Bemisia tabaci gen-nadius (Homoptera: Aleyrodidae). Int J Insect Morphol Em-bryol 24:297–306.

Rosell RC, Bedford ID, Frohlich DR, Gill RJ, Markham PG,Brown JK. 1997. Analyses of morphological variation in distinctpopulations of Bemisia tabaci. Ann Entomol Soc Am 90:575–589.

Rosell RC, Torres-Jerez I, Brown JK. 1999. Temporal pathway ofgeminivirus in whitefly extracts, saliva, hemolymph and hon-eydew. Phytopathology 89:239–246.

Salvucci ME, Rosell RC, Brown JK. 1998. Uptake and metabo-lism of leaf proteins by the silverleaf whitefly. Arch InsectBiochem Physiol 39:155–165.

Spurr AR. 1969. A low viscosity epoxy resin embedding mediumfor electron microscopy. J Ultrastruct Res 26:31–43.

van den Heuvel JFJM, Verbeek M, van der Wilk F. 1994. Endo-symbiotic bacteria associated with circulative transmission ofpotato leafroll virus by Myzus persicae. J Gen Virol 75:2559–2565.

van den Heuvel JFJM, Bruyere A, Hogenhout SA, Ziegler-GraffV, Brault V, Verbee M, van der Wilk F, Richards K. 1997. TheN-terminal region of the luteovirus readthrough domain deter-mines virus binding to Buchnera Gro-El and is essential forvirus persistence in the aphid. J Virol 71:7238–7265.

Wayadande AC, Baker GR, Fletcher J. 1997. Comparative ultra-structure of the salivary glands of two phytopathogen vectors,the beet leafhopper, Circulifer tenellus (Baker), and the cornleafhopper, Dalbulus maidis (Delong and Wolcott) (Homoptera:Cicadellidae). Inter J Insect Morphol Embryol 26:113–120.

Weber H. 1935. Der Bau der Imago der Aleurodinen, ein Beitrag-zur vergleichenden Morphologie des Insektenkorpers. Zoologica33:1–71.

Westcot DM, Ullman DE, Sherwood JL, Cantone FA, German TL.1993. Rapid fixation and embedding method for immunocyto-chemical studies of tomato spotted wilt tospovirus (TSWV) inplant and insect tissues. Microsc Res Tech 24:514–520.

Yokomi RK, Hoelmer KA, Osborne LS. 1990. Relationship be-tween the sweetpotato whitefly and the squash silverleaf dis-order. Phytopathology 80:895–900.

40 M. GHANIM ET AL.