The circulative pathway of begomoviruses in the whitefly vector Bemisia tabaci - insights from studies with Tomato yellow leaf curl virus

Ann appl Biol (2002) 140215-231Printed in Great Britain 215

Corresponding Author E-mail czosnekagrihujiacil

copy 2002 Association of Applied Biologists

The circulative pathway of begomoviruses in the whitefly vector Bemisia tabacimdash insights from studies with Tomato yellow leaf curl virus

By HENRYK CZOSNEK MIRIAM GHANIM and MURAD GHANIM

Institute of Plant Sciences and Genetics Faculty of Agriculture The Hebrew University of JerusalemRehovot 76100 Israel

(Accepted 1 May 2002 Received 23 October 2001)

Summary

Our current knowledge concerning the transmission of begomoviruses by the whitefly vector Bemisiatabaci is based mainly on research performed on the Tomato yellow leaf curl virus (TYLCV) complexand on a number of viruses originating from the Old World such as Tomato leaf curl virus and fromthe New World including Abutilon mosaic virus Tomato mottle virus and Squash leaf curl virus Inthis review we discuss the characteristics of acquisition transmission and retention of begomovirusesby the whitefly vector concentrating on the TYLCV complex based on both published and recentunpublished data

We describe the cells and organs encountered by begomoviruses in B tabaci We showimmunolocalisation of TYLCV to the B tabaci stylet food canal and to the proximal part of thedescending midgut and TYLCV-specific labelling was also associated with food in the lumen Themicrovilli and electron-dense material in the epithelial cells of the gut wall were also labelled by theanti TYLCV serum pointing to a possible virus translocation route through the gut wall and to aputative site of long-term virus storage We describe the path of begomoviruses in their vector Btabaci and in the non-vector whitefly Trialeurodes vaporariorum and we follow the rate of virustranslocation in these insects We discuss TYLCV transmission between B tabaci during matingprobably by exchange of haemolymph We show that following a short acquisition access to infectedtomato plants TYLCV remains associated with the B tabaci vector for weeks while the virus isundetectable after a few hours in the non-vector T vaporariorum The implications of the long-termassociation of TYLCV with B tabaci in the light of interactions of the begomovirus with insect receptorsare discussed

Key words Begomovirus tomato virus acquisition virus retention circulative transmission Bemisiatabaci Trialeurodes vaporariorum

Whitefly-Transmitted Geminiviruses

Geminiviruses are small plant virusescharacterised by a 22 nm acute 38 nm geminate particleconsisting of two joined incomplete icosahedraencapsidating circular single-stranded (ss) DNAgenome molecules of about 2700 nucleotides(Goodman 1977 Harrison et al 1977 Francki etal 1980 Zhang et al 2001) Geminivirusestransmitted by the whitefly Bemisia tabaci areassigned to the genus Begomovirus within the familyGeminiviridae (van Regenmortel et al 2000)Begomoviruses infect many important agriculturalplants worldwide including bean cassava cottonmelon pepper potato squash tobacco tomato andwatermelon Begomoviruses originating from theNew World have a bipartite genome organisationconsisting of two approx 28 kb circular ssDNAgenomic molecules named DNA A and DNA BBegomoviruses from the Old World have either a

DNA A-like monopartite genome or a bipartitegenome genetically similar to that of thebegomoviruses from the New World The genomesof monopartite viruses encode six genes two on thevirion genome strand (V1 and V2) and four on thecomplementary genome strand (C1 to C4) V1encodes the coat protein (CP) and V2 may controlsymptoms and movement C1 encodes the Repprotein necessary for virus replication C2 C3 areplication enhancer protein and C4 may affect host-range symptom severity and movement (Jupin etal 1994 Laufs et al 1995 Wartig et al 1997)The DNA A of bipartite viruses is similar inarrangement to the genome of monopartitebegomoviruses For New World begomoviruses theDNA A component lacks the V2 gene The DNA Bcomponent encodes BV1 and BC1 proteins that areessential for cell-to-cell and systemic movement(Noueiry et al 1994 Sanderfoot et al 1996) andcan influence host range (von Arnim amp Stanley

216 HENRYK CZOSNEK ET AL

1992 Ingham et al 1995) Although not directlyinvolved in interaction with the whitefly vectorDNA B sequences affect the efficiency of virusacquisition by the insect by determining the locationof begomoviruses in plant tissues (Liu et al 1997)

Co-Evolution of the Begomovirus-WhiteflyComplex

The begomovirus vector B tabaci is an insectspecies complex that has geographically distinctphenotypic and genotypic variants (Bird ampMaramorosch 1978 Perring et al 1993 Bedfordet al 1994 Brown et al 1995 Frohlich et al1999) The CP is the only begomoviral gene productthat directly interacts with whitefly factors duringthe circulat ive transmission of the virusPhylogenetic analysis of begomovirus CP sequencesresulted in the grouping of the viruses according totheir geographical origin 1) New World 2) WesternMediterranean basin 3) Middle East 4) Indiansubcontinent 5) East and Southeast Asia andAustralia (Rybicki 1994 Padidam et al 1995)Similarly the B tabaci complex could be resolvedinto five major groups based on mitochondrial DNAmarkers essentially coinciding with thegeographical distribution of the begomoviruses(Frohlich et al 1999 Brown 2001) This virus-vector co-adaptation is likely to be the result of co-evolution processes taking place in geographicallyisolated locations

Independent but converging information suggeststhat the whitefly-begomovirus interaction may belong-standing 1) Geminiviral DNA sequences seemto have integrated into the genome of some tobaccoancestors by illegitimate recombination duringNicotiana speciation about 25 million years ago(Bejarano et al 1996) 2) The endosymbioticbacteria that produce the GroEL homologuenecessary for the survival of begomoviruses in theirinsect vector (Morin et al 1999) have beenassociated with whiteflies for the last 200 millionyears (Baumann et al 1993) With the drift ofcontinents the initial whitefly-begomoviruscomplex(es) has developed with time intogeographically separated and co-adapted virus-insectcombinations (Bradeen et al 1997)

It is inevitable that during this long-lasting virus-vector relationship the virus has evolved to ensureboth its survival and efficient transmission by thewhitefly vector and the insect also has evolvedstrategies to safeguard it from possible deleteriouseffects of the virus Studying the interactions oftransmissible and non-transmissible begomoviruseswith vector and non-vector whitefly species mayhelp to identify the viral and cellular determinantsinvolved in transmission and shed some light on theevolutionary history of the begomovirus-whitefly

complex To this end we will discuss the associationof B tabaci with begomoviruses in particularTYLCV and related tomato infect ingbegomoviruses The nomenclature described byFauquet et al (2000) has been used to distinguishdistinct begomovirus species

Whitefly Cells and Organs Involved in theCirculative Transmission of Begomoviruses

In order to identify the position of receptors thatare likely to mediate circulative transmission ofbegomoviruses in their whitefly vector it isnecessary to describe in some detail the insect cellsand tissues involved The extensive anatomicalanalysis of the begomovirus non-vector whiteflyTrialeurodes vaporariorum performed in the 1930s(Weber 1935) still serves as a reference for analysingthe internal anatomy of whitefly species Severalrecent publications have focussed on the anatomyof B tabaci mouthparts (Rosell et al 1995) anterioralimentary canal (Hunter et al 1996) and digestivetract filter chamber and salivary glands (Cicero etal 1995 Harris et al 1995 1996 Ghanim et al2001a) Molecular studies have helped define thepathway of begomoviruses in their insect vector(Hunter et al 1998 Rosell et al 1999 Ghanim etal 2001b) Virus particles ingested through the Btabaci stylets enter the oesophagus and the digestivetract penetrate the gut membranes into thehaemolymph reach the salivary glands and finallyenter the salivary duct from where they are egestedwith the saliva A schematic drawing can be foundin Ghanim et al (2001b)

B tabaci feeds on phloem sap by inserting itsstylets into plant tissue and locating the vasculartissue (Pollard 1955) The stylet bundle is composedof three stylets the maxillary stylet which containsthe food canal (through which phloem is acquired)and the lateral salivary canal (through which salivais injected into the plant) and two mandibularystylets (Rosell et al 1995) The stylet food canalextends into the cibarium and oesophagus whichruns along the dorsal side of the thorax beforeentering the filter chamber The internal oesophagusexpands within the filter chamber where it is unitedwith the continuous lumen that extends into theconnecting chamber caecae descending andascending midguts Leaving the filter chamber thedescending midgut is composed of thick epithelialcells with large nuclei and microvilli extending intoa large lumen It is prolonged by the ascendingmidgut which narrows until it enters the filterchamber The ascending midgut is formed by verythick epithelial cells with an extensive brush borderof microvilli surrounding a rather small lumen Thehindgut terminates with the rectal sac (Ghanim etal 2001b)

217Whitefly transmission of begomoviruses

Cohen amp Nitzany 1966) TYLCV has an immenseeconomical impact worldwide (Picoacute et al 1996Nakhla amp Maxwell 1998) Molecular comparisonsof virus isolates from distinct geographical regionshave revealed that leaf curl disease of tomato iscaused by closely- as well as distantly-relatedmonopartite or bipartite begomoviruses (Czosnek ampLaterrot 1997)

Minimum time needed for efficient acquisition andinoculation of TYLCV by B tabaci

Whiteflies develop from an egg through fournymphal stages (also called instars) to an adult Btabaci instars are able to ingest and transmitbegomoviruses such as TYLCV (Cohen amp Nitzany1966) and Tomato yellow leaf curl Sardinia virus(TYLCSV) (Caciagli et al 1995) However thedisease is spread in the field by flying adults

Whitefly-mediated transmission of TYLCV totomato plants and observation of disease symptomshave indicated that the minimum acquisition accessperiod (AAP) and inoculation access period (IAP)were 15-30 min Moreover similar values wereobtained with TYLCV isolates from the Middle East(Ioannou 1985 Mansour amp Al-Musa 1992 Mehtaet al 1994) and from Italy (Caciagli et al 1995)and with Tomato leaf curl Bangalore virus(ToLCBV) isolates from India (Reddy ampYaraguntaiah 1981 Muniyappa et al 2000)However using PCR TYLCV DNA can be detectedin a single insect as early as 5-10 min after thebeginning of the AAP (Atzmon et al 1998 Ghanimet al 2001a Navot et al 1992) Similarly the viralDNA can be detected at the site of inoculation intomato after a 5 min IAP (Atzmon et al 1998)

A single insect is able to infect a tomato plant withTYLCV following a 24 h AAP although not allplants inoculated in this way will become infectedThe efficiency of transmission reaches 100 whenfive to 15 insects are used (Cohen amp Nitzany 1966Mansour amp Al-Musa 1992 Mehta et al 1994) Asimilar number of insects are necessary to achieve100 transmission of the New World bipartitegeminivirus Squash leaf curl virus (SLCV) (Cohenet al 1983)

Changes in acquisition and transmissionefficiency as a function of the age and the gender

of the insect vectorB tabaci reproduces by arrhenotoky Unfertilised

eggs give rise to haploid males while fertilised eggsdevelop into diploid females Mated females canregulate the sex of their progeny by selectivelyfertilising some of their eggs (reviewed by Byrne ampBellows 1991) The efficiency of TYLCVacquisition and transmission changes with the genderand age of B tabaci It has been reported previouslythat female whitefl ies transmit TYLCV and

The epithelial cells of the whitefly digestive tractseparate the gut lumen and the hemocoel whichoccupies the entire body cavity The hemocoelcontains the haemolymph or primitive blood systemwhich circulates around the body cavity between thevarious insect organs bathing them directly Itconsists of plasma in which are suspended severaltypes of nucleated cells or haematocytes andcontains various inorganic ions organic substancesand proteins An important function of thehaematocytes is phagocytosis of foreign proteinsmicroorganisms and tissue debris constituting a non-specific primitive immune system (Chapman 1991)Hence virions face a particularly hostileenvironment in the haemolymph

Endosymbiotic bacteria housed in the whiteflymycetocytes seem to have a cardinal role insafeguarding begomoviruses in the haemolymph Btabaci (biotype B) mycetomes contain two types ofendosymbionts the highly pleiomorphic P-type thatconstitutes approximately 80 of the totalpopulation and the coccoid C-type (Costa et al1995) The C-type endosymbionts produce a GroELhomologue that is released into the haemolymphbut not into the digestive tract (Morin et al 2000)As demonstrated for TYLCV the GroEL homologueseems to bind to and protect begomoviruses fromdegradation in the haemolymph (Morin et al 19992000)

A pair of primary salivary glands is located in theprothorax The paired accessory glands are muchsmaller and slightly anterior to the primary glandsThe primary salivary glands comprise at least 13nearly symmetrical large cells surrounding a centrallumen lined with microvilli which empties into aduct at the base of the gland This duct joins theaccessory salivary gland duct and the medial ductEach accessory gland is composed of four similarlarge cells that encircle a central lumen lined withextensive microvilli (Ghanim et al 2001b) Theprimary and accessory gland ducts on either sidefuse to form the lateral salivary ducts The two lateralducts fuse above the hypopharynx to form a singledual-channelled medial salivary duct (Harris et al1996) The salivary canal is contained almost entirelywithin one stylet while the food canal is centrallylocated and is formed by the apposition of the foodgrooves in both stylets The food and salivary canalsend at the stylet tip (Rosell et al 1995)

Parameters of Acquisition and Transmission ofTYLCV by the Whitefly Bemisia tabaci

Tomato yellow leaf curl virusTYLCV from Israel was one of the firs t

begomoviruses characterised in terms of itsrelationship with its vector the B biotype of Btabaci and its host range (Cohen amp Harpaz 1964


ToLCBV with higher efficiency than males (Cohenamp Nitzany 1966 Muniyappa et al 2000) In thesestudies the effect of age was not determined Wehave studied the effect of the gender and age ofsynchronised populations of adult B tabaci on theefficiency of transmission of TYLCV acquiredfollowing a 48 h AAP (Czosnek et al 2001) Nearlyall of the 1-2 wk-old adult females were able to causean infection in tomato plants following a 48 h IAPIn comparison only about 20 of the males of thesame age were able to produce infected plantsInoculation capacity decreased with the age of theinsects 60 of the 3 wk-old females were able tocause an infection in plants whereas no infectedplants were obtained following inoculation by malesof the same age Only 20 of the 6 wk-old femaleswere able to infect tomato plants Although the rateof TYLCV translocation is similar in males andfemales it is possible that different amounts of virustranslocate in the two genders (Ghanim et al2001a) and the putative begomovirus receptors inmales and females may differ In contrast femaleand male B tabaci transmitted SLCV with the sameefficiency (Polston et al 1990) The reason for thesedifferences is unclear

The decreased inoculation capability of ageingfemale whiteflies has been correlated with adiminution of the amount of TYLCV they acquireduring a 48 h AAP (Rubinstein amp Czosnek 1997)At the age of 17 days the insects acquired less thanhalf the virus acquired by 10 day-old insects and at24 days the amount was only about 10 At the ageof 28 days and thereafter the viral DNA associatedwith the insects was undetectable by Southern blothybridisation although the insects retained about20 of their initial capacity to produce infectedplants It is likely that being less active than youngwhiteflies in probing and feeding on infected plantsolder insects acquire fewer virus particles

Field and laboratory populations of B tabacicomprise males and females of various ages whichhave different abilities to acquire and transmitbegomoviruses The ratio of males to femaleschanges throughout the course of the year in the fieldas well as in the laboratory (Horowitz amp Gerling1992) Hence in our studies we generally use femaleB tabaci 1-2 wk after eclosion For practicalpurposes we suggest the use of synchronisedpopulations of insects of the same sex for studiesaimed at comparing parameters of acquisition andtransmission of B tabaci populations This isparticularly pertinent when whitefly-mediatedinoculation is the sole experimental tool to selecttomato genotypes with resistance or tolerance toTYLCV

Path of TYLCV in B tabaci and Speed of VirusTranslocation

Visualisation of TYLCV in sections of B tabaciVisualisation of begomoviruses in sections of

whiteflies may shed some light on the cells involvedin the translocation of virions and on the pathwaysthat have evolved to allow the crossing of the guthaemolymph and haemolymphsalivary glandbarriers Two bipartite begomoviruses (Tomatomottle virus ToMoV and Cabbage leaf curl virusCaLCuV) have been immunolocalised in the Btabaci filter chamber and in the anterior part of themidgut with ToMoV also detected in the salivaryglands (Hunter et al 1998)

We have initiated an extensive study of thelocalisation of TYLCV in anatomical sections ofviruliferous female B tabaci We have focused ourattention on those cells and organs involved in thecirculative transmission of this virus Using TYLCV-specific antiserum immunogold label was presentin the stylets (Fig 1) and was associated mainly withthe lumen of the food canal Label was detected inthe proximal part of the descending midgut (Fig 2)associated with food in the lumen and with electron-dense material in the microvilli-rich gut wallepithelial cells In another study we reported theimmunolocalisation of TYLCV to the filter chamberand the distal part of the descending midgut (Brownamp Czosnek 2002) These results suggest that themicrovilli may constitute one of the sites rich inbegomoviral receptors and may serve as the primarysite allowing internalisation of viral particles Hencethese cells may constitute a transit site for the viruson its way to the haemocoel or may serve as a viruslong-term storage site In another study we haveused in situ hybridisation to detect TYLCV in thenucleus of three of 14 of the cells of the B tabaciprimary salivary glands (Brown amp Czosnek 2002)Locating the virus in nuclei may suggest but doesnot prove replication of the virus within the insect

Speed of TYLCV translocation in B tabaciThe stylets of B tabaci must pass between the

epidermal and parenchymal cells before penetratingthe vascular tissues to allow the whitefly to feed inthe phloem (Costa 1969 Pollard 1955) Analysisof the electronic waveforms produced during feedingof B tabaci on Lima bean (Phaseolus lunatus) hasindicated that it took an average of 16 min (as earlyas 10 min for some of the insects as late as 45 minfor others) from initiation of leaf penetration tophloem ingestion (Walker amp Perring 1994) Theminimum phloem contact threshold period observedfor successful inoculation of TYLCV by B tabaciwas 18 min (Jiang et al 2000) PCR-based studiesof TYLCV ingestion and transmission have shownthat whiteflies may reach the phloem of tomato


plants within minutes after landing (Atzmon et al1998) Phloem probing occurs more quickly inTYLCV-infected tomato plants compared with non-infected plants Microscopic examination of infectedtomato leaves revealed that even before theappearance of symptoms spongy mesophyll cellscollapse leading to the displacement of the veinstowards the abaxial epidermis within closer reachof the whitefly stylets (Michelson et al 1997)

PCR has been a useful tool to determine the speedof begomovirus translocation in the whitefly vector(Caciagli amp Bosco 1997 Atzmon et al 1998 Rosellet al 1999 Ghanim et al 2001a) Temporal PCRanalysis of the translocation of the New World SLCVand the Old World TYLCV in tissues and organsinvolved in circulative transmission has shown thatthe timing of translocation is independent of theidentity of the virus (as long as it is transmissible)and of the geographical origin of the B tabaci vector

When DNA from B tabaci and salivahaemolymph and honeydew were used as substratesfor PCR SLCV DNA was detected in extracts of Btabaci after a 30 min AAP on infected pumpkin After2 h viral DNA was present in the haemolymphalthough it was detected in the saliva and honeydewonly after a further 6 h (Rosell et al 1999) We haveinvestigated the translocation of TYLCV DNA andCP using whitefly stylets head midguthaemolymph and salivary glands dissected from asingle insect (Ghanim et al 2001a) The organs andtissues were used directly as substrate for PCR andhomogenates were used in immunocapture-PCR (IC-PCR) TYLCV was detected in the head of whiteflies

as early as 10 min after the beginning of the AAPand in the midgut after approximately 40 min Thecrossing of TYLCV from the midgut to thehaemolymph was surprisingly fast virus reached thehaemolymph 30 min after it was first detected in themidgut just 90 min after the beginning of the AAPTYLCV was detected in the sal ivary glandsapproximately 55 h after it was first detected in thehaemolymph 7 h after the beginning of the AAPand approximately 1 h before the insects were ableto infect tomato plants The results obtained by PCRand by IC-PCR overlapped suggesting that the viralDNA is within virions These results showed thatonce acquired from infected plants begomovirusestransit in the body of B tabaci according to aninvariable sequential path head-midgut-haemolymph-salivary glands (Ghanim et al 2001a)

Translocation of TYLCV in T vaporariorum awhitefly species able to acquire but not to transmit

begomovirusesThe whitefly species T vaporariorum which also

feeds in the phloem and has a host range similar toB tabaci is capable of ingesting but not transmittingbegomoviruses such as SLCV (Polston et al 1990)and TYLCV (Antignus et al 1993) SLCV wasdetectable by PCR in whole body homogenates andhoneydew of T vaporariorum but not in thehaemolymph or saliva (Rosel l et al 1999)suggesting that the gut wall of the non-vectorwhitefly constitutes a barrier that begomoviruses areunable to cross To test this hypothesis we havefollowed concomitantly the translocation of TYLCV

Fig 1 Immunodetection of TYLCV in longitudinal sections through the stylets of a B tabaci female after a 24 h-acquisition access period on a TYLCV-infected tomato plant Insect heads were separated from the body and fixed in025 glutaraldehyde 4 paraformaldehyde in PBS for 3 h Following ethanol dehydration the tissues were infiltratedwith LR white resin and embedded in capsules (essentially as described by Wescot et al 1993) Sections of 60-90nm were deposited on 200 mesh formvar-coated nickel grids incubated for 3 h with a polyclonal antibody raisedagainst the TYLCV coat protein expressed in Escherichia coli (diluted 11000) followed by 1 h with a goat antirabbit IgG gold (15 nm diameter) conjugate stained with uranyl acetate and lead citrate and observed in a transmissionelectron microscope A section showing the food canal (FC) and the salivary canal (SC) (bar = 2 mm) B sectionshowing the food canal and the presence of TYLCV-specific labeling in the lumen (bar = 0 5 mm) C enlargementof boxed image in B showing the label in the lumen of the food canal


Fig 2 Immunodetection of TYLCV in longitudinal sections through the descending midgut of B tabaci femalesafter a 24 h acquisition access period on a TYLCV-infected tomato plant Dissected digestive tracts were washedwith PBS fixed and sections were processed as described in Fig 1 A Section through the descending midgut (Mvmicrovilli F food intake Lu lumen) note the label associated with food in the lumen (bar = 1 mm) B Sectionthrough the gut wall (Ec epithelial cell Gw gut wall) Insert enlargement of image boxed note the label associatedwith electron-dense material in the epithelial cell


in B tabaci and T vaporariorum from the time theinsects accessed infected tomato plants usingwhitefly head midgut haemolymph and salivaryglands as substrate for PCR (Ghanim et al 2001a)Analyses of the PCR products (Fig 3) showed thatTYLCV had reached the head of B tabaci and Tvaporariorum 10 min after the beginning of the AAPAfter 1 h the virus was found in the midgut of bothwhitefly species TYLCV DNA was detected in the

haemolymph of B tabaci after 2 h and in the salivaryglands after 8 h In contrast the virus was not foundin the haemolymph or the salivary glands of Tvaporariorum even after a 24 h AAP InterestinglyTYLCV was detected in a small number of midgutsamples These experiments indicate that TYLCVdoes not cross the guthaemolymph barrier of Tvaporariorum They may also suggest that most ofthe virus is destroyed in the digestive tract Hence

Haemolymph

Midgut

Head

Salivary glands

Bemisia tabaci Trialeurodesvaporariorum

Acquisition access Acquisition access

Min MinHours Hours

M 10 30 1 2 4 8 12 24 0 M 10 30 1 2 4 8 12 24 0

Fig 3 Comparative analysis of translocation of TYLCV in the vector B tabaci and in the non-vector T vaporariorumB tabaci and T vaporariorum were caged with infected tomato plants After the acquisition access periods indicatedgroups of five insects were collected dissected and the pooled heads midgut haemolymph and salivary glands weresubjected to PCR using TYLCV-specific primers (Ghanim et al 2001a) The PCR products were subjected toagarose gel electrophoresis and stained with ethidium bromide Note that viral DNA was not found in the haemolymphand the salivary glands of the begomovirus non-vector T vaporariorum 0 non-viruliferous whitefly M 1 kbpladder molecular weight marker


the inability of T vaporariorum to inoculate tomatoplants is correlated with the inability of TYLCV totransit from the digestive tract to the haemolymph

Transmission during mating another route ofacquisition of TYLCV by B tabaci

TYLCV from Israel can be transmitted betweenwhiteflies in a sex-dependent manner in the absenceof any other source of virus (Ghanim amp Czosnek2000) TYLCV was transmitted from viruliferousmales to females and from viruliferous females tomales but not between insects of the same sexTransmission took place when insects were cagedin groups or in couples either in a feeding chamberor on TYLCV non-host cotton plants All evidenceindicates that TYLCV is transferred during sexualcontact Non-viruliferous whiteflies were unable toingest detectable amounts of TYLCV from artificialmedium used to feed viruliferous whiteflies rulingout the possibility that the virus was acquired fromthe diet Transmission of TYLCV was observed onlywhen males and females were caged together notwhen whiteflies were of the same gender TYLCVwas detected first in the haemolymph of the recipientinsects later in their head but never in their digestivetract (Ghanim et al 2001a)

Two conditions have to be met in order to observevirus transmission amongst whiteflies First theinsects need to mate second virus needs to be presentin the haemolymph of the donor insect The key roleof the haemolymph was demonstrated by cagingwhiteflies previously fed on Abutilon mosaic virus(AbMV)-infected abutilon plants AbMV is a non-transmittable begomovirus it can be ingested by Btabaci but it does not cross the gut wall into thehaemolymph (Morin et al 2000) When non-viruliferous B tabaci males were caged with femalesfed on AbMV-infected abutilon plants AbMV DNAwas not detected in the males Identical results wereobtained in the reciprocal mating scheme (HCzosnek and M Ghanim unpublished) These resultssuggest that sexual transmission of TYLCV occursby exchange of haemolymph during intercourse

Acquisition and Long-Term Storage of TYLCVin B tabaci and T vaporariorum

Latent period of TYLCV in B tabaciOnce ingested begomoviruses are not

immediately available for infection They need totranslocate from the digestive tract to the salivaryglands from which they are excreted with the salivaduring feeding The time it takes for a geminivirusto complete this path and to infect susceptible plantsis called the latent period The latent period may notonly reflect the speed of virus translocation but alsothe time it takes for an insect to accumulate enoughvirions (the number is undetermined) to be able to

efficiently transmit the disease to plants For somebegomoviruses this threshold may be reached muchearlier than for others For example SLCV has beendetected by PCR in the saliva 8 h after the beginningof the AAP (Rosell et al 1999) while the minimallatent period was reported to be approximately 19 h(Cohen et al 1983) In contrast TYLCV has beendetected in the salivary glands of B tabaci 7 h afterthe beginning of the AAP only 1 h before the insectswere able to transmit virus to produce infectedtomato plants (Ghanim et al 2001b) The estimatedlatent period for a given virus may vary due to theexperimental conditions or to changes in virus andor vector with time For example the latent periodof TYLCV from Israel was reported to be 21 h inthe early 1960s (Cohen amp Nitzany 1966) while itwas found to be 8 h 35 years later (Ghanim et al2001b) Clearly care is needed when making suchcomparisons

Whiteflies acquire a finite amount of TYLCVduring a feeding episode

Begomoviral DNA in B tabaci accumulates withincreasing AAP on infected plants up to a peak atapproximately 12 h for TYLCV (Zeidan amp Czosnek1991) 24 h for TYLCSV (Caciagli amp Bosco 1997)and 48 h for SLCV (Polston et al 1990) At thepeak the insects contained the equivalent ofapproximately 600 million viral genomes (about 1ng viral DNA) It seems therefore that the amountof virions an insect can acquire from an infectedplant during a single feeding event is finite reachinga steady state between ingestion and egestion after12-48 h of AAP

We have designed an experiment to determinewhether consecutive feedings lead to thedisplacement of the acquired virus (Fig 4)Whiteflies were first caged with tomato plantsinfected with TYLCV for 48 h Then the insectswere collected and caged with tomato plants infectedwith TYLCSV for an additional 48 h Quantificationof the viral DNAs showed that as TYLCSVaccumulated during the second feeding the amountof TYLCV remained approximately constant Atthe end of the two successive 48 h AAPs thewhiteflies contained approximately similar amountsof TYLCV and TYLCSV These results showed thatthe newly acquired virus did not chase the virusalready associated with the insect At the end of thesuccessive AAP the tomato plants infected by thesewhiteflies contained similar amounts of TYLCV andTYLCSV DNA These results contrast with earlierexperiments showing that whiteflies that were fedfor 48 h on SLCV-infected squash then transferredto Melon leaf curl virus (MLCuV)-infectedwatermelon for 24 h exhibited a 35-90 reductionin transmission of MLCuV during a 48 h IAPcompared with those fed on MLCuV only (Cohen


amp Bosco 1997)In most instances the viral DNA remained

associated with the insects for much longer thantransmission ability suggested For example whileTYLCSV DNA was detectable up to 20 days afterthe end of the 48 h AAP transmission could occuronly for up to 8 days (Caciagli et al 1995) Detectionof viral DNA (by Southern blot hybridisation orPCR) and CP (by Western blot immunodetection orIC-PCR) suggests these are not retained in B tabacifor the same time periods Following the end of the48 h AAP TYLCV DNA was detected throughoutthe 5 wk life span of the insect while the amount ofTYLCV CP steadily decreased until it wasundetectable at day 12 (Rubinstein amp Czosnek1997) The disappearance of the virus CP was

et al 1989) The results were interpreted as aninterference of transmission of MLCuV by SLCVHowever the transmission of SLCV by whitefliesharbouring the SLCV-MLCuV virus mixture was notassessed and the amount of virus acquired duringthe successive AAP was not measured

Retention of TYLCV in B tabaci and Tvaporariorum

Following a 1-2 day AAP begomoviruses may beretained in their whitefly vector for several weeksand sometimes for the entire life of the insect SLCVand TYLCV remain associated with B tabaci duringthe entire life of the vector (Cohen et al 1989Rubinstein amp Czosnek 1997) while TYLCSV isundetectable after approximately 20 days (Caciagli

100

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0 10 20 30 40 50 60 70 80 900

Acquisition access feeding (h)

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LC

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LC

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TYLCV

Fig 4 Successive acquisition of TYLCV and TYLCSV by B tabaci Whiteflies were caged with a tomato plantinfected with TYLCV Groups of 20 whiteflies were collected every 2-4 h After 48 h the remaining insects werecollected and caged with a tomato plant infected with TYLCSV Groups of 20 whiteflies were removed every 2-4 hTotal DNA was extracted from all the groups of 20 insects and DNA equivalent to one whitefly per time point wereSouthern blotted The samples were hybridised with a radiolabelled TYLCV probe (Navot et al 1991) washed athigh stringency (to discriminate between the two viruses) and exposed to X-ray film The TYLCV probe was removedand the samples were hybridised with a radiolabeled TYLCSV probe (Kheyr-Pour et al 1991) washed at highstringency and exposed to X-ray film The autoradiograms were scanned and the DNA quantified using cloned viralDNA standards (1 pg DNA is equivalent to 06 million genomes) Vertical arrows indicate the beginning of AAP ofthe two viruses Horizontal arrows point to the autoradiograms obtained after hybridisation with the virus-specificprobes Note that during the second feeding whiteflies acquired amounts of TYLCSV similar to the amounts ofTYLCV acquired during the first feeding The quantities of TYLCV remained approximately constant during theacquisition of TYLCSV


infected tomato plants (Fig 6) TYLCV DNA wasdetected in B tabaci over the entire 7 days of theexperiment while the CP was detected during thefirst 4 days only In contrast TYLCV DNA wasdetected in T vaporariorum only during the first 6h that followed the end of the AAP and the CP forup to 4 h Thus TYLCV vanished very quickly fromT vaporariorum once acquisition feeding hasceased but nonetheless the DNA appears to beretained longer than the CP even in the non-vector

Reduced longevity and fertility of viruliferous Btabaci and TYLCV invasion of the insect

reproductive systemThe life-long association of TYLCV with B tabaci

led to a significant decrease of the insect longevityMortality curves of whiteflies reared on eggplants

associated with a rapid decrease in the ability of thewhitefly to produce infected host plants as shownfor TYLCV (Rubinstein amp Czosnek 1997) andSLCV (Cohen et al 1983) It is interesting to notethat a difference in the retention of viral DNA andCP in B tabaci was also observed with an Israeliisolate of the non-transmissible bipart itebegomovirus AbMV (Morin et al 2000) Followinga 4-day AAP on infected abutilon plants the virusDNA remained associated with B tabaci throughoutthe 15 days sampling period while the CP wasdetectable only for up to 7 days (Fig 5)

TYLCV was retained for much shorter time in thenon-vector T vaporariorum than in the B tabacivector We have compared the retention periods ofTYLCV DNA and CP in the two insect speciesreared on cotton for 7 days following a 3 h AAP on

DNA(Hybridisation)

CP(IC-PCR)

B

Days after acquisition access

1 2 1 2 1 2 1 2 1 2

P 0 3 5 8 12 15

0 4 7 10P

14

Fig 5 Retention of AbMV in B tabaci Whiteflies were transferred to cotton plants following a 4-day access toinfected abutilon plants During the 15 day experiment three groups each of 20 insects were collected at the timepoints indicated DNA was prepared from insects of the first group and divided into two equal portions which wereSouthern blot hybridised respectively with radiolabelled probes for AbMV DNA A (A) and AbMV DNA B (B)(Frischmuth et al 1990) Extracts from the second and third groups of 20 insects each (1 and 2) were incubated withPCR tubes coated with an antiserum raised against the CP of Tomato golden mosaic virus (which recognises the CPof AbMV) the DNA from the immunocaptured virions was detected by PCR with primers specific to AbMV DNAA (Morin et al 2000) The PCR products were subjected to agarose gel electrophoresis and stained with ethidiumbromide P infected abutilon plant Note that DNA A and B were detected during the entire experiment while the CPwas detectable by immunocapture-PCR (IC-PCR) only up to 7 days after the end of the AAP

A


(a TYLCV non-host plant) were established after 1day-old adult female acquired TYLCV from infectedtomato for a 48 h AAP The difference at the 50mortality point between viruliferous and non-viruliferous whiteflies was between 5 and 7 daysThus the association of TYLCV with B tabaci ledto a reduction of 17-23 in the whitefly lifeexpectancy compared with insects that have notacquired the virus (Rubinstein amp Czosnek 1997)Similarly the life span of female whiteflies fed for

24 h on SLCV-infected plants was on average 25shorter than that of whiteflies fed on the same virussource for 4 h (Cohen et al 1983) Theseobservations indicate that at least these twobegomoviruses have deleterious effects on theirinsect vector

The long-term association of B tabaci withTYLCV also affected the insect fertility (Rubinsteinamp Czosnek 1997) The effect was not immediateas if the virus had first to invade the reproductive

Hours Days

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

Hours Days

Time after acquisition access

Fig 6 Retention of TYLCV in B tabaci and in T vaporariorum Whiteflies of the two species were caged withinfected tomato plants After 3 h of access feeding the insects were removed and transferred to cotton plants aTYLCV non-host plant At the time points indicated two groups of 10 insects each were collected the first groupwas used to assess the presence of TYLCV DNA by PCR the second group was used to determine the presence ofthe virus coat protein (CP) by immunocapture PCR (IC-PCR) The 10 whiteflies from the first group were pooledtheir DNA extracted and subjected to PCR with TYLCV-specific primers The 10 whiteflies from the second groupwere pooled and insect extracts were incubated with PCR tubes coated with an antiserum raised against the TYLCVCP the DNA from the immunocaptured virions was detected by PCR with TYLCV-specific primers (Ghanim ampCzosnek 2000) The PCR products were subjected to agarose gel electrophoresis and stained with ethidium bromideNote that TYLCV DNA was detected in B tabaci over 7 days while the CP was detected during the first 4 days onlyIn contrast TYLCV DNA was detected in T vaporariorum only during the first 6 h while the CP was found for up to4 h

Trialeurodesvaporariorum

DNA(PCR)

CP(IC-PCR)

DNA(PCR)

CP(IC-PCR)

Bemisiatabaci


system During the 24 h following the AAP the meannumber of eggs laid by viruliferous and non-viruliferous insects was similar (60 vs 51) Themean number of eggs laid by 3 day-old viruliferousinsects during a 7 day or a 20 day period wassignificantly lower than that laid by non-viruliferousinsects (227 vs 381 and 560 vs 334 respectively)Eggs maturing in the ovaries of viruliferous B tabacicontained viral DNA detectable by PCR indicatingthat TYLCV has invaded the reproductive systemThus infection of the maturing egg may have led toits abortion Following oviposition viral DNA wasdetected in some but not all eggs instars and adultprogeny of individual whiteflies A relatively smalland variable proportion of insects that developedfrom eggs of viruliferous whiteflies were able totransmit the virus to tomato in biological tests(Ghanim et al 1998) In another study using adifferent B tabaci colony and an almost identicalTYLCV isolate viral DNA was detected in eggs andup to the third instar but not in the adult progeny ofviruliferous insects In contrast in the sameinvestigation ToMoV was not found in eggs andinstars and was not transmitted to progeny (Polstonet al 2001)

Viral and insect Determinants of BegomovirusCirculative Transmission

Viral determinantsIt is believed but not proven that begomoviruses

are acquired translocated and inoculated as virusparticles by the whitefly vector It is therefore likelythat the capsid is the structure that is exposed to thewhitefly tissues and interacts with insect receptorsand chaperones This hypothesis is supported byexperiments in which acquisition of Bean goldenmosaic virus by B tabaci was lost followingmutagenesis of the CP gene (Azzam et al 1994)Furthermore replacing the CP of AbMV with thatof the closely-related whitefly transmissible Sidagolden mosaic virus (SiGMV) produced atransmissible chimeric AbMV (Houmlfer et al 1997)Vector specificity of geminiviruses is determined bythe CP and there is no evidence for the involvementof other virus-encoded proteins in transmissionExchanging the CP gene of the whitefly-transmittedAfrican cassava mosaic virus (ACMV) with that ofthe leafhopper-transmitted Beet curly top virus(BCTV genus Curtovirus) produced a leafhopper-transmitted ACMV chimera (Briddon et al 1990)

Loss of transmission by B tabaci can be causedby a surprisingly small number of amino acidreplacements in the begomovirus CP NaturalTYLCSV mutants have been isolated which areacquired but not transmitted by B tabaci althoughthey are able to systemically infect tomato plantsfollowing agroinoculation Loss of transmission was

due to the replacement of two amino acids atpositions 129 and 134 in the TYLCSV CP (Noris etal 1998) This region of the CP is also implicatedin transmission of the bipartite Watermelon chloroticstunt virus (Kheyr-Pour et al 2000) AbMV isanother begomovirus that has lost the ability to betransmitted (Wu et al 1996) probably because ithas been maintained and propagated by cuttingsMutagenesis of AbMV CP showed that exchange oftwo amino acids at CP positions 124 and 149 weresufficient to restore partial transmissibility bywhiteflies Alteration of amino acid 174 in additionto those at position 124 and 149 completely restoredtransmission of AbMV (Houmlhnle et al 2001)

Insect determinantsThe mechanism of transmission of viruses of the

genera Tospovirus (family Bunyaviridae) andLuteovirus (Luteoviridae) show many similaritiesto that of the geminiviruses (Nault 1997) Theseviruses are transmitted in a propagative manner bythrips and a circulative manner by aphidsrespectively Investigations into the transmission ofthese viruses is more advanced than those ongeminiviruses and thus point the way for study ofgeminivirus transmission These systems haveindicated the involvement of two sites regulatingvirus transmission 1) the gut epithelia in the thriptospovirus (Ullman et al 1992) and aphidluteovirus(Gildow 1993) systems 2) the basal lamina and thebasal plasmalemma of the accessory salivary glandin the aphidluteovirus system (Peiffer et al 1997Gildow et al 2000) In the begomovirusB tabacisystem the digestive tract was the only organ shownto be a barrier to transmission of some virusesAbMV as well as some non-transmissible TYLCSVmutants are unable to cross the midgut membranesinto the haemolymph These mutants may have lostthe capsid structure enabling binding to putativereceptors in the vector midgut followed bytranslocation to the haemolymph

It is likely that begomovirus receptors may bepresent in the midgut of B tabaci Indeed the midgut(within and outside the filter chamber) and internalileum epithelia have a brush border at the apicalmembrane and the microvilli would provide an idealsite for viral endocytosis (Fig 2 Ghanim et al2001b) In contrast the oesophagus caecumcontinuous lumen within the filter chamber andrectum are lined with a cuticular intima making themunlikely sites of virus uptake In the tospovirusthripsystem a receptor has been isolated from theplasmalemma of the thrip midgut which may servein virus attachment (Bandla et al 1998)

Comparison of vector and non-vector insects hasallowed the isolation of virus receptor candidatesTwo proteins isolated from head tissues of the aphidvector Sitobion avenae but not from the non-vector


aphid Rhopalosiphum maidis have been identifiedas potential receptors for Barley yellow dwarfluteovirus (Li et al 2001) Similarly it might bepossible to isolate begomoviral receptors bycomparing protein profiles of the midgut from thevector B tabaci and the non-vector T vaporariorumThe receptor properties may be confirmed byprotein-protein interaction studies using virusoverlay assays and the yeast two-hybrid system

Endosymbionts appear to play a cardinal role inthe safe transit of begomoviruses within their insectvectors The role of chaperonins synthesised byinsect endosymbiotic bacteria was first demonstratedin aphids where an interact ion between theluteovirus and the endosymbiotic chaperone GroELwas shown to be essential for virus retention (vanden Heuvel et al 1994) The survival of TYLCVand probably other begomoviruses in thehaemolymph of B tabaci is likely to be ensured bya similar strategy GroEL produced by the whiteflycoccoid bacterium was identified in the insecthaemolymph as a native 14-mer unit each subunithaving a mass of 63 kDa (Morin et al 2000)TYLCV particles displayed affinity for the B tabaciGroEL homologue in a virus overlay assay and theTYLCV CP and B tabaci GroEL interacted in theyeast two-hybrid system Interestingly B tabaciGroEL interacted as well with the CP of the non-transmissible AbMV (Morin et al 2000) indicatingthat mutations in the CP which prevented AbMVfrom crossing into the insect haemolymph (Houmlhnleet al 2001) do not prevent binding to GroEL

The function of GroEL in the circulativetransmission of begomoviruses may not be limitedto protection in the haemolymph We do not knowhow begomoviruses penetrate and cross the gutepithelial cells We do not know whether the virustranslocates as a geminate particle and whether itchanges its conformation in the process In the lattercase the role of GroEL might be to correctly refoldthe viral particle once in the haemolymph

Concluding Remarks

Begomovirus-whitefly co-adaptationPlant viruses and especially geminiviruses have

much more specific relationships with their insectvector than with plant hosts (reviewed by Power2000) The evolutionary constraints that narrowedthe geminivirus-vector complex to a onebegomovirus-one insect species are not understoodCapsid structure and insect receptors are likely tobe the key to this one-to-one relationship

Evolution of begomoviruses might have beentowards a better adaptation of the capsid to putativereceptors of the local whitefly to ensure optimisationof virus transmission Hence a begomovirusinfecting a given host tends to possess a CP with

epitopes more closely related to those of otherbegomoviruses in the same geographical region thanto a virus infecting the same host in other regionsFor example the CP of Indian cassava mosaic virusis more similar to those of other geminiviruses ofthe Indian subcontinent than to ACMV Similarlytransmission of TLCV by an insect from the samegeographical region is more efficient than when thevirus and insect originate from two different regions(McGrath amp Harrison 1995)

Recombination as a driving force in the rapidevolution of geminiviruses has been appraisedrecently (Padidam et al 1999 Harrison amp Robinson1999) Although it seems that recombination in theCP gene is less frequent than in other parts of thegeminiviral genome in those viruses where CPrecombination has occurred the transmissiondeterminants dictated by the amino acid stretchesthat influence the structure of the virus CP have beenpreserved and possibly improved (Sanz et al 1999)

As the begomoviral capsid evolved toward a betteradaptation of the virus to the insect some whiteflyspecies may have developed receptors in theirdigestive and salivary systems that facilitatebegomovirus translocation The question remainswhy whiteflies have evolved a system that allowsthe circulative transmission of potentially harmfulbegomoviruses instead of confining the virus to thestylet or destroying the virus in the digestive systemIndeed available data suggest that TYLCV as wellas SLCV are reminiscent of insect pathogens andare deleterious to their whitefly vector (Cohen etal 1983 Rubinstein amp Czosnek 1997) Thewhitefly host may have developed antiviral strategiesthrough the expression of factors preventing virusreplication as described in the case of TYLCV(Cohen 1967 Cohen amp Marco 1970)

Chaperonins produced by the vectorendosymbionts may be part of the overall strategydevised to neutralise deleterious viruses It has beensuggested that viruses belonging to unrelatedtaxonomic groups have taken advantage ofendosymbiotic bacterial proteins produced by theirinsect vector to avoid degradation in thehaemolymph (Gibbs 1999) However it is possiblethat the purpose of translocating viruses throughoutthe insect body is ultimately to remove potentiallyharmful particles Accordingly facing invasion byprogenitors of modern begomoviruses whiteflyancestors like aphids (van den Heuvel et al 1994)may have taken advantage of chaperoninssynthesised by endosymbionts (Baumann et al1993) to facilitate the transit of the virions until theyare expelled instead of attempting to destroy themby expressing enzymes or antiviral factors Thisstrategy may not have been adopted by the whiteflyT vaporariorum which is able to ingest but nottransmit begomoviruses (Rosell et al 1999) Once


ingested during feeding the virus spreads in thedigestive tract of T vaporariorum but is unable tocross the gut epithelial cells into the haemolymphand is destroyed within hours (Fig 6)

Long-term association of begomoviruses with thewhitefly vector

B tabaci ingests increasing quantities ofbegomovirus during feeding on an infected plantuntil the amount of virions present in the insect bodyreaches an ingestion-egestion steady state after 12-48 h of AAP Following the end of the AAP the viralDNA remains detectable for much longer than theCP sometimes during the entire life of the insect(Rubinstein amp Czosnek 1997) Since only aninfinitesimal amount of virus is egested duringfeeding and excreted with the honeydew thequestion as to the fate of the ingested virus isintriguing The finite number of putativebegomoviral receptors may become saturated duringthe first hours of acquisition feeding We postulatethat with longer AAP the virions that do not interactwith the receptors leave the circulative pathway andinvade as yet unidentified cells and tissues wherethey are stored In the process the virionsdisassemble the viral DNA binds to proteins thatprotect it from degradat ion and the CP isprogressively destroyed Once acquisition accessends the supply of virion ceases The particles boundto the insect receptors progressively leave thedigestive tract reach the haemolymph and thesalivary glands and may be transmitted to plantsThis process may continue during the entire adultlife of B tabaci explaining the residual infectivityof TYLCV observed 4 wk after the end of the AAP(Rubinstein amp Czosnek 1997) In contrast wesuppose that when the non-vector T vaporariorumaccesses infected plants the ingested virions do notencounter begomoviral receptors in the insect gutand therefore are not retained in the digestive tractof this insect for more than a few hours (Fig 6)

The invasion-storage hypothesis may besubstantiated by an experiment where B tabaciacquires successively two begomoviruses TYLCVfollowed by TYLCSV While the kinetics ofTYLCSV acquisition was similar to that of TYLCVthe amount of TYLCV acquired during the firstfeeding remained approximately constant (Fig 4)If B tabaci does not possess a different set ofreceptors for TYLCV and TYLCSV the results maybe explained by the progressive displacement ofTYLCV by TYLCSV According to this hypothesisTYLCSV binds to the B tabaci receptors previouslyoccupied by TYLCV and most of the TYLCVinvades unidentified insect tissues and leaves thecirculative pathway although enough TYLCVremains in the digestive tract to allow co-infectionof tomato plants along with TYLCSV

The pattern of long-term association of the non-transmissible AbMV with B tabaci also may beinterpretated in the light of the invasion-storagehypothesis We speculate that following acquisitionAbMV binds to the putative B tabaci receptorspresent in part of the digestive tract Howeverbecause of a change in the structure of the capsiddue to mutations in the CP (Wu et al 1996 Houmlhnleet al 2001) AbMV cannot be internalised in theepithelial cells by the microvilli system and deliveredto the haemolymph With time the virions may leavethe receptors invade insect tissues in which the viralgenome is protected and the CP progressivelydestroyed (Fig 6)

The persistence of begomoviruses in B tabaci asinfective entities for longer than the latent periodsometimes for the entire life of the insect raises thequestion of replication of the virus in the insectCurrently it is postulated that geminiviruses do notreplicate in their insect vectors (Harrison 1985)However data showing accumulation of TYLCVDNA in B tabaci reared on a TYLCV non-host plantafter first feeding on a TYLCV-infected plantsuggested multiplication of TYLCV in its vector(Mehta et al 1994) Begomovirus DNA acquiredover 24-48 h remains associated with the insect forseveral weeks much longer than infectivity (Caciagliamp Bosco 1997 Rubinstein amp Czosnek 1997Muniyappa et al 2000) Persistence of the viralDNA may suggest a certain level of replication orsome association beyond what is expected of acirculative transmission model Long-term retentionof TYLCV negative effects on longevity andfecundity of the host (Rubinstein amp Czosnek 1997)and transmission to eggs (Ghanim et al 1998Polston et al 2001) supports this hypothesis sincedeleterious effects on the insect and transovarialtransmission are all characteristics associated withreplication of a plant virus in its insect vector(Sylvester amp Richardson 1969 Sylvester 1973Gingery 1988) The recently developed continuouswhitefly cell line originating from B tabaciembryonic tissues might provide a tool to studyputative begomovirus replication and expression(Hunter amp Polston 2001)

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Kheyr-Pour A Bendahmane M Matzeit V Accotto G PCrespi S Gronenborn B 1991 Tomato yellow leaf curlvirus from Sardinia is a whitefly-transmitted monopartitegeminivirus Nucleic Acids Research 196763-6769

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1992 Ingham et al 1995) Although not directlyinvolved in interaction with the whitefly vectorDNA B sequences affect the efficiency of virusacquisition by the insect by determining the locationof begomoviruses in plant tissues (Liu et al 1997)

Co-Evolution of the Begomovirus-WhiteflyComplex

The begomovirus vector B tabaci is an insectspecies complex that has geographically distinctphenotypic and genotypic variants (Bird ampMaramorosch 1978 Perring et al 1993 Bedfordet al 1994 Brown et al 1995 Frohlich et al1999) The CP is the only begomoviral gene productthat directly interacts with whitefly factors duringthe circulat ive transmission of the virusPhylogenetic analysis of begomovirus CP sequencesresulted in the grouping of the viruses according totheir geographical origin 1) New World 2) WesternMediterranean basin 3) Middle East 4) Indiansubcontinent 5) East and Southeast Asia andAustralia (Rybicki 1994 Padidam et al 1995)Similarly the B tabaci complex could be resolvedinto five major groups based on mitochondrial DNAmarkers essentially coinciding with thegeographical distribution of the begomoviruses(Frohlich et al 1999 Brown 2001) This virus-vector co-adaptation is likely to be the result of co-evolution processes taking place in geographicallyisolated locations

Independent but converging information suggeststhat the whitefly-begomovirus interaction may belong-standing 1) Geminiviral DNA sequences seemto have integrated into the genome of some tobaccoancestors by illegitimate recombination duringNicotiana speciation about 25 million years ago(Bejarano et al 1996) 2) The endosymbioticbacteria that produce the GroEL homologuenecessary for the survival of begomoviruses in theirinsect vector (Morin et al 1999) have beenassociated with whiteflies for the last 200 millionyears (Baumann et al 1993) With the drift ofcontinents the initial whitefly-begomoviruscomplex(es) has developed with time intogeographically separated and co-adapted virus-insectcombinations (Bradeen et al 1997)

It is inevitable that during this long-lasting virus-vector relationship the virus has evolved to ensureboth its survival and efficient transmission by thewhitefly vector and the insect also has evolvedstrategies to safeguard it from possible deleteriouseffects of the virus Studying the interactions oftransmissible and non-transmissible begomoviruseswith vector and non-vector whitefly species mayhelp to identify the viral and cellular determinantsinvolved in transmission and shed some light on theevolutionary history of the begomovirus-whitefly

complex To this end we will discuss the associationof B tabaci with begomoviruses in particularTYLCV and related tomato infect ingbegomoviruses The nomenclature described byFauquet et al (2000) has been used to distinguishdistinct begomovirus species

Whitefly Cells and Organs Involved in theCirculative Transmission of Begomoviruses

In order to identify the position of receptors thatare likely to mediate circulative transmission ofbegomoviruses in their whitefly vector it isnecessary to describe in some detail the insect cellsand tissues involved The extensive anatomicalanalysis of the begomovirus non-vector whiteflyTrialeurodes vaporariorum performed in the 1930s(Weber 1935) still serves as a reference for analysingthe internal anatomy of whitefly species Severalrecent publications have focussed on the anatomyof B tabaci mouthparts (Rosell et al 1995) anterioralimentary canal (Hunter et al 1996) and digestivetract filter chamber and salivary glands (Cicero etal 1995 Harris et al 1995 1996 Ghanim et al2001a) Molecular studies have helped define thepathway of begomoviruses in their insect vector(Hunter et al 1998 Rosell et al 1999 Ghanim etal 2001b) Virus particles ingested through the Btabaci stylets enter the oesophagus and the digestivetract penetrate the gut membranes into thehaemolymph reach the salivary glands and finallyenter the salivary duct from where they are egestedwith the saliva A schematic drawing can be foundin Ghanim et al (2001b)

B tabaci feeds on phloem sap by inserting itsstylets into plant tissue and locating the vasculartissue (Pollard 1955) The stylet bundle is composedof three stylets the maxillary stylet which containsthe food canal (through which phloem is acquired)and the lateral salivary canal (through which salivais injected into the plant) and two mandibularystylets (Rosell et al 1995) The stylet food canalextends into the cibarium and oesophagus whichruns along the dorsal side of the thorax beforeentering the filter chamber The internal oesophagusexpands within the filter chamber where it is unitedwith the continuous lumen that extends into theconnecting chamber caecae descending andascending midguts Leaving the filter chamber thedescending midgut is composed of thick epithelialcells with large nuclei and microvilli extending intoa large lumen It is prolonged by the ascendingmidgut which narrows until it enters the filterchamber The ascending midgut is formed by verythick epithelial cells with an extensive brush borderof microvilli surrounding a rather small lumen Thehindgut terminates with the rectal sac (Ghanim etal 2001b)








































Haemolymph

Midgut

Head

Salivary glands



Min MinHours Hours

M 10 30 1 2 4 8 12 24 0 M 10 30 1 2 4 8 12 24 0




















100

Mil

lion

gen

omes

200

300

400

500

600

700

0 10 20 30 40 50 60 70 80 900


TY

LC

V

TY

LC

SV

TYLCSV

TYLCV









DNA(Hybridisation)

CP(IC-PCR)

B


1 2 1 2 1 2 1 2 1 2

P 0 3 5 8 12 15

0 4 7 10P

14


A





Hours Days

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

Hours Days




DNA(PCR)

CP(IC-PCR)

DNA(PCR)

CP(IC-PCR)

Bemisiatabaci
















Concluding Remarks















References






















































































































































Haemolymph

Midgut

Head

Salivary glands



Min MinHours Hours

M 10 30 1 2 4 8 12 24 0 M 10 30 1 2 4 8 12 24 0




















100

Mil

lion

gen

omes

200

300

400

500

600

700

0 10 20 30 40 50 60 70 80 900


TY

LC

V

TY

LC

SV

TYLCSV

TYLCV









DNA(Hybridisation)

CP(IC-PCR)

B


1 2 1 2 1 2 1 2 1 2

P 0 3 5 8 12 15

0 4 7 10P

14


A





Hours Days

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

Hours Days




DNA(PCR)

CP(IC-PCR)

DNA(PCR)

CP(IC-PCR)

Bemisiatabaci
















Concluding Remarks















References







































































































































Haemolymph

Midgut

Head

Salivary glands



Min MinHours Hours

M 10 30 1 2 4 8 12 24 0 M 10 30 1 2 4 8 12 24 0




















100

Mil

lion

gen

omes

200

300

400

500

600

700

0 10 20 30 40 50 60 70 80 900


TY

LC

V

TY

LC

SV

TYLCSV

TYLCV









DNA(Hybridisation)

CP(IC-PCR)

B


1 2 1 2 1 2 1 2 1 2

P 0 3 5 8 12 15

0 4 7 10P

14


A





Hours Days

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

Hours Days




DNA(PCR)

CP(IC-PCR)

DNA(PCR)

CP(IC-PCR)

Bemisiatabaci
















Concluding Remarks















References





























































































































Haemolymph

Midgut

Head

Salivary glands



Min MinHours Hours

M 10 30 1 2 4 8 12 24 0 M 10 30 1 2 4 8 12 24 0




















100

Mil

lion

gen

omes

200

300

400

500

600

700

0 10 20 30 40 50 60 70 80 900


TY

LC

V

TY

LC

SV

TYLCSV

TYLCV









DNA(Hybridisation)

CP(IC-PCR)

B


1 2 1 2 1 2 1 2 1 2

P 0 3 5 8 12 15

0 4 7 10P

14


A





Hours Days

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

Hours Days




DNA(PCR)

CP(IC-PCR)

DNA(PCR)

CP(IC-PCR)

Bemisiatabaci
















Concluding Remarks















References




















































































































Haemolymph

Midgut

Head

Salivary glands



Min MinHours Hours

M 10 30 1 2 4 8 12 24 0 M 10 30 1 2 4 8 12 24 0




















100

Mil

lion

gen

omes

200

300

400

500

600

700

0 10 20 30 40 50 60 70 80 900


TY

LC

V

TY

LC

SV

TYLCSV

TYLCV









DNA(Hybridisation)

CP(IC-PCR)

B


1 2 1 2 1 2 1 2 1 2

P 0 3 5 8 12 15

0 4 7 10P

14


A





Hours Days

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

Hours Days




DNA(PCR)

CP(IC-PCR)

DNA(PCR)

CP(IC-PCR)

Bemisiatabaci
















Concluding Remarks















References


















































































































Haemolymph

Midgut

Head

Salivary glands



Min MinHours Hours

M 10 30 1 2 4 8 12 24 0 M 10 30 1 2 4 8 12 24 0




















100

Mil

lion

gen

omes

200

300

400

500

600

700

0 10 20 30 40 50 60 70 80 900


TY

LC

V

TY

LC

SV

TYLCSV

TYLCV









DNA(Hybridisation)

CP(IC-PCR)

B


1 2 1 2 1 2 1 2 1 2

P 0 3 5 8 12 15

0 4 7 10P

14


A





Hours Days

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

Hours Days




DNA(PCR)

CP(IC-PCR)

DNA(PCR)

CP(IC-PCR)

Bemisiatabaci
















Concluding Remarks















References

































































































































100

Mil

lion

gen

omes

200

300

400

500

600

700

0 10 20 30 40 50 60 70 80 900


TY

LC

V

TY

LC

SV

TYLCSV

TYLCV









DNA(Hybridisation)

CP(IC-PCR)

B


1 2 1 2 1 2 1 2 1 2

P 0 3 5 8 12 15

0 4 7 10P

14


A





Hours Days

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

Hours Days




DNA(PCR)

CP(IC-PCR)

DNA(PCR)

CP(IC-PCR)

Bemisiatabaci
















Concluding Remarks















References





















































































































100

Mil

lion

gen

omes

200

300

400

500

600

700

0 10 20 30 40 50 60 70 80 900


TY

LC

V

TY

LC

SV

TYLCSV

TYLCV









DNA(Hybridisation)

CP(IC-PCR)

B


1 2 1 2 1 2 1 2 1 2

P 0 3 5 8 12 15

0 4 7 10P

14


A





Hours Days

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

Hours Days




DNA(PCR)

CP(IC-PCR)

DNA(PCR)

CP(IC-PCR)

Bemisiatabaci
















Concluding Remarks















References






















































































































DNA(Hybridisation)

CP(IC-PCR)

B


1 2 1 2 1 2 1 2 1 2

P 0 3 5 8 12 15

0 4 7 10P

14


A





Hours Days

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

Hours Days




DNA(PCR)

CP(IC-PCR)

DNA(PCR)

CP(IC-PCR)

Bemisiatabaci
















Concluding Remarks















References



















































































































Hours Days

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

M P 1 2 4 6 8 12 1 2 3 4 5 6 7

Hours Days




DNA(PCR)

CP(IC-PCR)

DNA(PCR)

CP(IC-PCR)

Bemisiatabaci
















Concluding Remarks















References






























































































































Concluding Remarks















References



















































































































Concluding Remarks















References






















































































































References















































































































































































































































































































Documents

The circulative pathway of begomoviruses in the whitefly vector Bemisia tabaci - insights from studies with Tomato yellow leaf curl virus