Zoomorphology (1995) 115 : 31-39 �9 Springer-Verlag 1995

J. M. Cicero �9 E. Hiebert - S. E. Webb

The alimentary canal of Bemisia tabaci and Trialeurodes abutilonea (Homoptera, Sternorrhynchi}: histology, ultrastructure and correlations to function

Accepted: 23 August 1994

Summary The midguts of Bemisia tabaci and Tria- leurodes abutilonea are looped so that the anterior and posterior extremities are in contact with each other. The basal lamina is breached at this point so that opposing basal epithelial membranes of either extremity are con- tiguous. On the anal side of the contact is a "filter-or- gan", consisting of oesophageal, Malpighian and modi- fied ventricular epithelial cells. Comparison with model systems indicates that the filter-organ acts as an os- moregulatory device. It appears that fluid food is direct- ed into the looped midgut and concentrated by passive transport of water across the contact point, through the filter-organ, and then into the hindgut. The filter-organ of both species is attached to the contact point, but in T. abutilonea it is thereafter suspended in the alimentary lumen, free of the alimentary wall. In B. tabaci, the oesophageal cells of the filter-organ are attached to the alimentary wall. This constitutes the major difference in gut histology between the two species. Dissections indi- cate that the midgut can be moved through the petiole, from the abdomen to the thorax, and back again. This, and the absence of Malpighian tubules, suggests that the midgut services the excretory needs of the flight muscles.

Introduction

The sweet potato whitefly, Bemisia tabaci Gennadius, 1889, is a vector for several geminiviruses that are re- sponsible for devastating annual crop losses (Brown and Bird 1992). Persistent transmission of plant viruses by insects generally involves the digestive system (Co- hen and Antignus 1994; Gildow 1985; Nault 1989). The

J. M. Cicero ([2]) �9 E. Hiebert Department of Plant Pathology, University of Florida, Gainesville 32611, U S A

S. E. Webb CFREC Leesburg, University of Florida, Gainesville 32611, USA

need for an understanding of the alimentary canal ultra- structure is, therefore, essential.

The whitefly digestive system has been studied at the organ level by several authors (Chudhry and Gupta 1970; Goodchild 1966; Mahmood 1955; Singh 1949; Weber 1933, 1935). All describe a long, thin oesophagus that bears paired salivary glands anteriorly and spans the thorax to pass through the petiole and connect with the midgut. The midgut descends to the posterior and then ascends back so that both extremities are located in the base of the abdomen. Two prominent diverticula occur at one of these ends and a short convolution oc- curs at the other. There is confusion as to which end the oesophagus connects, and, therefore, disagreement as to the direction of fluid flow, as well as whether the diver- ticula are caecal or Malpighian. The two midgut ex- tremities are somehow attached to each other in this region. A narrow ileum and wider rectum follow the convolution.

Chundry and Gupta and the other formentioned au- thors believe that water can pass through this attach- ment, from the anterior midgut directly into the hindgut, so as to concentrate nutrients in the midgut and avoid dilution of the haemolymph. Although none present supporting evidence, precedents for such a shunt are widespread among Homoptera. The simplest involve a twisting together of mid- and hindgut. The most elaborate, known from model Cicadoidea, are re- ferred to as filter chambers. The model chfimbers have a sheath that encloses the two ventricular extremities and the bases of the Malpighian tubules in one house so that ionic gradients can be established to mediate the neces- sary osmotic interactions (Gouranton 1968). Weber (1935) and Goodchild (1966) believe a filter chamber occurs inside the whitefly convolution.

In this paper we examine the' oesophagus, midgut and hindgut of B. tabaci and the non-vector Tri- aleurodes abutilonea (Haldeman, 1850), as a preliminary to employment of techniques for determining pathways of viral transmission (Gildow 1982; Gildow and Gray 1993).

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A

D OE

AS / / ~

IL ~ N

RE / ADE FO COP M

Fig. 1A-D. Whitefly midgut. A Habitus. Ventriculus is in abdo- men, caeca are in thorax. B Habitus. Ventriculus and caeca are in thorax. Not noted for Trialeurodes abutilonea. In Bemisia tabaci, both conditions occur irrespective of gravidity. C Midgut, ventral view. D Close-up of boxed area in C. Convolution is straightened. AM, PM anterior- and posteriormost positions of a confine (CO) noted for attachment of posterior ventriculus to anterior ven- triculus, AN to anus, AS ascending ventriculus, CA right caecum, DE decrease in diameter of ascending ventriculus, DS descending ventriculus, FO filter organ, IL ileum, MTMalpighian tissue, OE external oesophagus, PR pronotum; RE rectum, SA spherical anal structure

Materials and methods

I. Extirpation

Adult females were glued, venter down, to stiff paper and pinned to wax-bottomed watch glasses filled with phosphate-buffered sa- line. Under 100X magnification, a Nanopoint diamond scalpel (Microstar, Houston, Texas) was passed blindly about the interior of the thorax to sever the oesophagus. The abdominal cuticle was then slit anteriorly and peeled back so that the haemolymph could be flushed out with a micropipette and toluidine blue introduced to lightly stain the organs. Canals with their midgut situated in the thorax were rejected. The canal was severed free from the anus and put into ice-cold fixative (2.5 % glutaraldehyde, 2% formalde- hyde, pH 7.4). Specimens were fixed overnight at 2-3 ~ C, rinsed in buffer, osmicated, rinsed again, dehydrated in ethanol and then embedded in Spurr's medium.

tions of B. tabaci, two intact T. abutilonea and three intact B. tabaci. Unless otherwise stated, descriptions refer to both whitefly species.

Results

I. Gross construct ion

Herein, the two large diverticula will be referred to as caeca (Fig. 1A, CA), identifying the ventricular extremi- ty to which they are at tached as the anter ior extremity. "Midgut" will refer to ventriculus + caeca. At the poste- r ior end of the ventriculus, inside the convolution, there occurs a "fi l ter-organ" which receives and incorporates the oesophagus with a layer of cells comparab le to those of Malpighian tubules in the model systems (Fig. 1D, MT]. The oesophageal and Malpighian tissues have lu- mina of their own, therefore, the lumen that occurs f rom the midgut through to the hindgut will be termed the "cont inuous lumen". The poster ior extremity is at- tached to the anter ior extremity by a gateway (Fig. 2A, G A ) whose posit ion is variable between individuals within a short confine (Fig. 1D, CO).

II. Haemocoel ic or ientat ion

II. Intact abdomens

Adult female thoraces were checked in buffer for the midgut with toluidine blue. Abdomens were severed above the petiole with the scalpel and fixed as above. After rinsing in ice-cold buffer, osmica- tion was allowed to proceed at 2-3 ~ C until the anterior half of the abdominal cavity turned black. After dehydration as above, and embedding in LR Gold at - 2 0 ~ C, the abdomens were oriented for an anterior approach to the microtomy knife. Separation be- tween the epicuticle and plastic was rectified by applying a minute amount of cyanoacrylate glue to the fissure as an alternative to gamma-glycidoxypropyl trimethoxysilane (Lindley 1992). The blocks were allowed to dry thoroughly to avoid the formation of a monolayer on the boat water,

III. Numbers

About 75 adult females of both species combined were dissected and taken to various levels of elucidation. Fine details were based primarily on two extirpations of T. abutilonea, four extirpations of

Affixed to each other, the two extremities of the ven- triculus are held in the base of the a b d o m e n because of opposed lengths of the oesophagus, which passes through the petiole, and the hindgut, which extends to the anus. Otherwise, the posi t ion of the midgut is highly variable (Fig. 1A, B). Unconnected to other organs, it is capable of rota t ing abou t the oesophagus-hindgut axis and squeezing th rough the petiole into the thorax. Dis- sections show that different individuals can have their midgut disposed through the petiole to different extents. In some cases, all aspects are confined to the abdomen. In other cases, one or bo th caeca occur in the thorax while the ventriculus is disposed in ~the abdomen. The midgut was observed to be totally disposed in the tho- rax of some gravid females of B. tabaci but not others, and has not yet been observed in T. abutilonea. When in this condition, the caeca and ventriculus can extend to

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Fig. 2 A T. abutilonea. Right-facing view of extirpated alimentary canal, embedded and photographed through the block face. Hair- pin turn (arrows) of the convolution is foremost in focus. Dark region within the convolution is the filter organ. The B. tabaci canal is of the same design. B--D B. tabaci. B Long section from extirpated canal of site where oesophagus penetrates into the con- volution. C Cross-section of oesophagus from extirpated canal near site of penetration. D Ventriculus. Arrows point to basal

involutions. AP apex of filter organ, AS ascending ventriculus, DS descending ventriculus and right caecum (out of focus), GA gate- way, I L ileum, I N iutima-lined lumen of oesophagus, L U lumen of midgut, OE oesophagus penetrates through left face of convolu- tion, IO internal oesophageal lumen turns to right, towards anus, on entering the convolution and proceeds into interior of the filter-organ, PL pleats in sheath, RE rectum, SC sheath of convo- lution, SO sheath of oesophagus, U N unidentified

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Fig. 3 B. tabaci. Cross-section Of convolution. ATattachment of filter-organ to convolution's epithelium (CE), BA basal lamina arising from ascending ventriculus and encircling filter organ, CO continuous lumen, H A haemocoel, I N intima-lined lumen of in- ternal oesophagus, L A lateral membranes of oesophageal cells, M C Malpighian cells, M L Malpighian lumen, M M modified ven- tricular cell sharing basal lamina with Malpighian cell (note polygonal involutions), M O same, sharing basal lamina with oesophageal cell (note lack of polygonal infoldings), OC oeso- phageal cells, SH sheath

the cervical area where they might easily be confused with the accessory salivary glands, which also show mi- crovilli in cross-section (in preparation). With all midgut sections in their "resting state", untwisted and unfolded, and the caeca pos i t ioned left and right, the ventriculus descends from the caeca along the dorsal abdominal interior and ascends ventrally.

III. Configurat ion of the convolut ion

A tunica or other ensheathing membrane was not found associated with the convolution. In cases of extreme dis-

placement of the midgut (Fig. 1B) the convolution is pulled straight. Otherwise, the configuration of the rest- ing state is as follows. After the gateway, the canal bends sharply to the right before entering into a hairpin turn (Fig. 2A). After the hairpin, the canal then bends sharply to the left, then to the posterior as the ileum.

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IV. Sheath

The alimentary canal consists primarily of two epithe- lial layers, an inner, luminal layer and an outer sheath (Fig. 2B, SO, SC; Fig. 3, SH). The sheath appears to be continuous on the oesophagus and hindgut, but discon- tinuous on the midgut. These cells present a basal lami- na, ca. 0.24 gm thick, on both their apical and basal surfaces. The basal lamina continues to invest areas of the luminal midgut epithelium where sheath cells are lacking so that all aspects of the canal in contact with the blood are lined. Bundles of muscle fibres occur out- side and inside the cells of this sheath.

V. Oesophagus

All sections were taken from the abdominal end and, therefore, the extent to which the profiles in Fig. 2B, C are represented in the thorax and head is unknown. The oesophagus is about 1/15 the diameter of the ventriculus in buffer-dissected specimens. It is composed of an inti- ma-lined luminal epithelium invested by the sheath. It penetrates the left face of the convolution at a point distad of the gateway (Figs. 1D, 2A). There is apparently some variation in the location of this site.

In B. tabaci, the sheath elaborates into pleats (Fig. 2C, PL) jus t before the oesophagus penetrates into the convolution. The muscle bands characteristic of the sheath are specifically associated with these pleats. These pleats were not located in T. abutilonea. In both species, the luminal epithelium contains a granular body of unknown composition (Fig. 2C, UN). This body and the lumen can be traced down into the convo- lution. No aspect of the sheath was seen to enter inside.

VI. Midgut

The ventriculus is slightly inflated at the caecal junc- tions (Fig. 1C) and decreased in diameter just before the gateway (Fig. 1D, DE). A constriction in diameter was noticed at about the centre of the ascending arm, other- wise the midgut is uniform in diameter. No changes in histological nor luminal continuity were noted. Caecum and ventriculus could not be readily distinguished from each other in sections of intact specimens.

The luminal epithelia of these two organs are com- posed entirely of brush-border cells. Replacement cells were not observed. The basal membranes are involuted into the cytoplasm as open lamellae and, perhaps,

Fig. 4 A B. tabaci. Convolution interior, distad of the distal ex- tent of the oesophageal lumen. B T. abutiIonea. Long section of convolution. BA basal lamina, CE convolution's epithelium, CO continuous lumen, IN intima-lined lumen of internal oesophagus, MI microvilli of Malpighian lumen, MVmodified ventricular ep- ithelium, OC oesophageal cell, OE oesophagus, PL pleat, note associated ribbon (RI), PO polygonal basal infoldings

closed compartments (Fig. 2D). There is no indication that these compartments are a separate cell layer as the cytoplasm and organelles are continuous in and out of them. The brush-border cells are very large and bear large nuclei and numerous lysosomes. Malpighian tubules were not found outside the alimentary canal.

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Fig. 5A-E Aspects of whitefly anatomy. A B. tabaci extirpation. Gateway between anterior ventriculus (AI/) and Malpighian cells (MC) of the convolution. In this photograph, opposing sheath cells (SH) on the right side of the gateway are pressed tightly together so that no haemocoel (HA) is present. B T abutilonea. Unknown structure attached to the exterior of the hairpin. C T abutiIonea extirpated ileum. D B. tabaci rectum. CO continuous lumen, CR crystalline inclusion, IN intima-lined lumen

VII. Convolution

The filter-organ is sac-like in shape, affixed to the gate- way basally and extends into the continuous lumen for about three-quarters of the length of the convolution (Fig. 2A). It attenuates in diameter to a one-cell tip. It consists of two inner layers of cells surrounded by one outer layer (Fig. 3) and there are no intervening sinuses between them. The basal membranes of the two inner layers are apposed to the basal membranes of the outer layer across a basal lamina (Fig. 3, BA). The basal mem- branes of the two inner layers do not meet. One of the inner layers is the internalized oesophagus with its inti- ma-lined lumen. The other, also possessing a lumen,

brush-bordered in this case, resides exclusively in the filter-organ and will be referred to as Malpighian. Both of these lumina appear to be closed; indications of con- tinuity between them and between the continuous lu- men were specifically looked for but not found.

The outer layer and the basal lamina are continua- tions of the epithelium of the ascending ventriculus and will be called a "modified ventricular epithelium". Cells of this epithelium that are contiguous with Malpighian cells (Fig. 3, MM) have a brush-bordered apical mem- brane and more or less symmetrical, polygonal infold- ings along the basal membrane. The Malpighian cells present polygonal infoldings in their basal membranes as well.

On entering the convolution, the oesophageal cells are directed to the right (Fig. 2B, IO) and end before the filter-organ's apex (Fig. 4A). Their basal membranes do not present polygonal infoldings and their lateral mem- branes interrupt the brush border in areas where they are open to the Malpighian lumen (Fig. 3, LA). More- over, the modified ventricular cells discontinue their in- foldings and brush border where they contact the oesophageal cells (Fig. 3, MO).

The ventricular epithelium ceases to line the outer wall of the continuous lumen when it contacts the filter organ. Another epithelium, referred to as the "convolu- tion's epithelium" (Fig. 3, CE), characterized by apical membranes thrown into ribbons (Fig. 4A, RI), begins at this point and lines the continuous lumen until the latter exits the convolution as the hindgut. The modified ven- tricular epithelium is pleated in several places and the ribbons are sometimes included in them.

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sheath surrounds the rectum externally (Fig. 5D). In cross-section, the apices of rectal epithelial cells appear as clavate extensions into the lumen. A spherical, possi- bly glandular, structure occurs near the anus (Fig. 1A, SA).

Discussion

I. Cicadoid model

VIII. Gateway

The anterior and posterior ventricular extremities ad- here because of a breach in the continuity of the basal lamina, leaving a gateway ca. 7.0 gm in cross-sectional width, wherein the basal membranes of the cells in the anterior extremity and the basal membranes of the Malpighian cells are in direct contact. The gatewa}r could be located only in extirpated specimens. In intact abdomens, organs are pressed tightly together such that opposed flanks of sheath cells (Fig. 5A, SH) could not be recognized. None of our sections showed luminal conti- nuity across this gateway.

IX. Differences between species

The entry of the oesophagus into the convolution was not determined for T. abutilonea. Distad of this point, however, specimens of T. abutilonea showed no indica- tion that the filter-organ is attached to the convolution's epithelium. Rather, it is suspended in the convolution, free of the convolution's epithelium for its entire length (Fig. 4B). In B. tabaci, the filter-organ is attached to the convolution's epithelium. The cells that mediate this at- tachment (Fig. 3, AT) could not be identified with cer- tainty. They may originate from the external oesophageal epithelium, the convolution's epithelium or they may be a new cell type. All the cells involved in this attachment undergo complex intercalations with basal laminae and have yet to be resolved.

A minute structure (Fig. 5B) appears to be attached to the outside of the convolution in T. abutilonea only. It is wedged in the inner fold of the hairpin and so may or may not occur there naturally. It is composed of at least two cells showing a high volume to volume ratio of mitochondria, rough endoplasmic reticula and dense, laminar crystals.

Cheung and Marshall (1973a, b) and Marshall and Che- ung (1974) studied sections of cicadoid filter chambers in order to infer osmoregulatory capacity from the ar- rangement of membranes within and their reaction to various cytochemical assays.

They provide a schematic (see Fig. 2 in Marshall and Cheung 1974) showing that the filter chamber is a dila- tion of an alimentary segment immediately following the oesophagus. A so-called conical segment and midgut follow and no caeca are mentioned. The conical segment has not been homologized. The filter chamber consists of, from exterior to interior, a sheath, an outer lumen and the "filter chamber proper" with its own lu- men. Their descriptions imply that the latter is the con- tinuous lumen. The midgut loops back to the anterior and its posterior extremity passes through the sheath into the outer lumen. The posterior extremity coils about the outer surface of the filter chamber proper, then produces Malpighian tubules before exiting through the sheath as the hindgut. The tubules travel down the outer lumen, exit through the sheath and pro- ceed to the rectum. All three, the internalized midgut, internalized tubules and filter chamber proper, are free of each other. Each presents strongly infolded basal membranes towards each other across the outer lumen, and microvilli to their respective inner lumina.

They make no mention of whether oesophageal cells participate in the filter chamber, however, their figures do not indicate that an intima occurs in the interior. They suggest that fluid flows from the oesophagus into the filter chamber proper and down to the conical seg- ment. According to their indirect assays, osmoregula- tion results in passive water transport through the walls of the filter chamber proper, into the outer lumen and thence into the internalized tubes for disposal through the hindgut.

II. Proposed model for the whitefly

X. Hindgut

The ileum (Fig. 5C) is a narrow tube consisting of a continuous sheath and an intima-lined luminal epitheli- um. The latter contains numerous mitochondria and vesicles. The proximal extent of the intima was not lo- cated. It could occur at the distal end of the convolution, since no intima was located within. A thisk muscular

Fluid moves down through the oesophagus and proba- bly collects in the pleated, apparently contractile, crop (Fig. 2C; see Deshpande 1933, Plate IX, Fig. 2, Cp). From there it passes into the oesophageal lumen of the filter organ. The location of the distal end of the oesoph- agus, within the filter-organ, is puzzling. Since our in- vestigation did not determine whether or not direct con- tinuity actually exists between the oesophageal lumen

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Fig. 6A, B Model proposed for direction of fluid flow. A Sphinc- ter (small arrows) closes the ileum so that fluid flow (large arrow) is diverted into the ventriculus. B Subsequently, sphincter closes over filter organ and water is passed through the gateway to the hindgut

and the continuous lumen, any speculation of function is premature. However, a model for the direction of fluid flow can be built using areas of the canal that might be constricted by sphincter muscles (Fig. 6).

Fluid passing out of the filter-organ and into the con- tinuous lumen of the convolution must then be diverted to the midgut. Sphincter muscles would accomplish this by closing the access to the hindgut (Fig. 6A). After the midgut is filled, the hindgut sphincter would open and the muscles enveloping the convolution would contract over the filter-organ so that the continuous lumen of the convolution is closed. Water would then be eliminated through the filter-organ, by way of the gateway, and into the hindgut (Fig. 6B). The role of the filter-organ, therefore, would be to maintain the osmotic pressure necessary for this transport.

Alternatively, water and sap might be separated im- mediately on entry into the filter organ, with water di- rected to the hindgut and sap through the gateway. In either case, we suggest that the midgut serves as a reser- voir, storing large amounts of sap during filtration.

The epithelium of the cicadoid filter chamber proper is probably a continuation of the ventricular epithelium. Its osmoregulatory function is to hold back solutes while allowing water to pass into the outer lumen. If the whitefly model is correct, it would be the functional ana- logue of the epithelial cells on the ventricular side of the gateway. Functionally, at least, the filter-organ would then correspond to the cicadoid internal midgut and internal Malpighian tubules. Since the outer layer of the filter-organ is modified ventricular epithelium, it is rea- sonable to refer to the inner layer as Malpighian. The only major difference between the two systems is that the cicadoid internal tubes are free of the filter chamber proper, while in the whitefly, the basal membranes of the two adhere. The cicadoid sheath is comparable in posi- tion and cytostructure to the convolution's epithelium. In both insect groups, the oesophagus breaches this en- sheathment.

Marshall (1983) used frozen sections of the cicada, Cyclochila australasiae Don, for X-ray microanalysis to confirm their earlier contention (Marshall and Cheung 1974) that the outer lumen is actually a blood sinus.

There is no indication that haemolymph is present in- side the whitefly convolution.

III. Polarity of the whitefly midgut

Weber (1933, 1935) recognized that the oesophagus is connected to the convolution and labelled this point as the anterior extremity of the ventriculus, as is typical in insects. Correspondingly, he labelled the diverticula as Malpighian tubules. Interpretations of the other au- thors are variously incompatible with each other and with our findings in many ways.

Arguably, polarity of the insect midgut might be de- fined by attachment to the oesophagus and hindgut, by the direction of fluid flow or by the physiological roles of its component sections. Establishing polarity for the whitefly midgut must be tentative because of conflicts that occur between interpretations based on these three paradigms and because there is no reason to give prefer- ence to one of these criteria over any other. In fact, Cheung and Marshall (1973b) demonstrate that al- though sharing a continuous lumen, different sections of the midgut and Malpighian tubes (ducts + tubules) have different metabolic roles. Anterior and posterior midgut sections are involved in different osmoregulato- ry tasks while the middle section is involved in storage excretion. In the whitefly, the constriction in diameter, noticed at about the centre of the ascending arm may demarcate ventricular subsections (see Cheung and Marshall 1973a, p 652) or may be an artifact of han- dling. Since the whitefly midgut is the only organ able to directly access the flight muscles, it is highly likely that some aspect of it plays a major role in the uptake of metabolic wastes. Waste would be expelled by reopen- ing the convolution's sphincter and voiding the midgut through the continuous lumen. The functions of nutri- ent concentration and waste collection may go on alter- nately. Complete translocation of the midgut into the thorax may limit flight and modify other behaviours.

Acknowledgements We wish to extend our appreciation to Dr. Avas Hamon and John Heppner of the State of Florida Depart- ment of Agriculture and Consumer Service's Division of Plant Industry, and to the University of Florida's Interdisciplinary Core for Biological Research, Drs. Dan Purcifull and Henry Aldrich for extensive use of their facilities and instruments. Drs. Henry Aldrich and James Nation gave critical review to the manuscript. This research was supported by Grant No. IS-1813-90 from BARD, the United States-Israel Binational Agricultural Research and Development Fund. Florida Agricultural experiment station journal series paper R-04150.

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