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Transovarial transmission of Rickettsia and organ-specific infection of the whitefly Bemisia 1

tabaci 2

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Marina Brumin1, Maggie Levy2 and Murad Ghanim1# 4

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1Department of Entomology, the Volcani Center, Bet Dagan 50250, Israel and 2 Department of 6

Plant Pathology and Microbiology, Robert H. Smith Faculty of Agriculture, Hebrew University 7

of Jerusalem, Rehovot 76100, Israel. 8

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#Correspondence: M. Ghanim, E-mail: ghanim@agri.gov.il 11

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Running title: Rickettsia – Whitefly interactions 14

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01184-12 AEM Accepts, published online ahead of print on 1 June 2012

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The whitefly Bemisia tabaci is a cosmopolitan insect pest that harbors Portiera 24

aleyrodidarum, the primary obligatory symbiotic bacterium, and several facultative 25

secondary symbionts. Secondary symbionts in B. tabaci are generally associated with the 26

bacteriome, ensuring their vertical transmission; however, Rickettsia is an exception and 27

occupies most of the body cavity, except the bacteriome. The mode of Rickettsia transfer 28

between generations and its subcellular localization in insect organs were not investigated. 29

Using electron and fluorescence microscopy, we show that Rickettsia infects the digestive, 30

salivary and reproductive organs of the insect, however, it was not observed in the 31

bacteriome. Rickettsia invades the oocytes during early developmental stages and resides in 32

follicular cells and cytoplasm; it is mostly excluded when the egg matures, however, some 33

bacterial cells remain in the egg, ensuring their transfer to subsequent generations. 34

Rickettsia was localized to testicles and the spermatheca, suggesting a horizontal transfer 35

between males and females during mating. The bacterium was further observed at high 36

amounts in midgut cells, concentrating in vacuolar-like structures, and was located in the 37

hemolymph, specifically at exceptionally high amounts around bacteriocytes and in fat 38

bodies. Further infected organs by Rickettsia included the primary salivary glands and 39

stylets, sites of possible secretion of the bacterium outside the whitefly body. The close 40

association between Rickettsia and B. tabaci digestive system might be important for 41

digestive purposes. The vertical transmission of Rickettsia to subsequent generations occurs 42

via the oocyte and not, like other secondary symbionts, the bacteriome. 43

44

45

46

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INTRODUCTION 48

The intimate interactions between endosymbionts and their arthropod hosts are well established 49

for obligate primary symbionts such as Buchnera in aphids and Carsonella in psyllids (3, 27). 50

These bacteria are housed within specialized cells termed bacteriocytes and aggregate together to 51

form an organ termed bacteriome (3, 12, 56). Primary endosymbionts provide the host with 52

essential amino acids to complete their diet, and are therefore necessary for host survival and 53

development (12). In contrast, secondary symbionts are not essential and until recently, their 54

effect on their host's biology was underestimated. In the past few years however, their relevance 55

has become a matter of interest and accumulating data suggest that they can play a significant 56

role in the biology of their hosts (56). To date, secondary symbionts have been reported to be 57

involved in reproductive manipulations, host plant utilization, and ability to cope with 58

environmental factors such as response to heat stress and chemical insecticides (8, 11, 43, 46, 49, 59

57, 58). The reported localization patterns of secondary symbionts are diverse and vary within 60

the host body. The bacteria can be either diffusely distributed throughout the entire host body or 61

restricted to specific tissues. For example, secondary symbionts have been reported from the 62

hemolymph of many insect taxa (15, 28, 34, 67), primary bacteriocytes in whiteflies (39, 63), 63

secondary bacteriocytes and sheath cells mainly in aphids (34, 55, 61, 67), salivary glands (54, 64

59, 60), Malpighian tubules (13) and reproductive organs (28, 32, 41, 60, 68). 65

Like other phloem-feeders, the sweetpotato whitefly, Bemisia tabaci (Gennadius) 66

(Hemiptera: Aleyrodidae), harbors a diverse array of endosymbionts, including the primary 67

endosymbiont Portiera aleyrodidarum (3), and several other facultative secondary symbionts, 68

including Rickettsia, Hamiltonella, Wolbachia, Arsenophonus, Cardinium, and Fritschea (3, 38). 69

B. tabaci is a complex of several cryptic species, also termed biotypes, which differ genetically 70

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and biologically. The most widespread and damaging biotypes are B and Q [recently termed 71

Middle East Asia Minor 1 (MEAM1) and Mediterranean (MED), respectively] (24). The B 72

biotype from Israel has been associated with Hamitonella, while the Q biotype is associated with 73

Wolbachia and Arsenophonus. Both biotypes harbor Rickettsia with infection rates ranging from 74

22 to 100% (17). Neither biotype harbors Cardinium or Fritschea (17). A molecular 75

phylogenetic analysis based on 16S rDNA and gltA sequences revealed that Rickettsia sp. from 76

B. tabaci belongs to the group consisting of Rickettsia bellii from ticks and Rickettsia spp. from 77

herbivorous arthropods such as aphid and leafhopper. This Rickettsia shares 99 and 97% 78

similarity with R. bellii 16S rDNA and gltA sequences, respectively (38). Rickettsia symbionts 79

have been further identified from a wide variety of invertebrates such as aphids (15), leafhoppers 80

(22), lady bird beetles (69), bruchid beetles (33), leeches (47) and ticks (6). 81

All secondary symbionts in B. tabaci co-localize with the primary symbiont inside the 82

bacteriocytes, ensuring their vertical transmission. However, only Rickettsia localizes outside the 83

bacteriocytes and appears in most of the body cavity except the bacteriocytes (39). In only one 84

case, Rickettsia was described co-localizing with the primary symbiont inside the bacteriocyte 85

(14, 39). It is still unclear how Rickettsia that localizes outside the bacteriosome is transferred 86

and spread in B. tabaci populations, especially that it can reach high prevalence and near fixation 87

in natural populations as was recently shown in Arizona (45). The later study has further shown 88

that Rickettsia-infected B. tabaci females exhibit high fitness benefits such as increased 89

fecundity, a greater rate of survival, and host reproduction manipulation via the production of a 90

higher proportion of daughters (45). Additional studies have shown that the presence of 91

Rickettsia in B. tabaci populations influenced the whitefly’s response to heat stress by benefitting 92

its host under high temperatures (11), and has also shown to increase the whitefly's susceptibility 93

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to chemical insecticides (49). Studies on Rickettsia from other invertebrates have revealed 94

diverse effects of the bacteria on their hosts. For example, in the pea aphid Acyrthosiphon pisum, 95

Rickettsia-infected individuals showed lower fresh body weight, reduced fecundity and 96

significantly suppressed densities of Buchnera, suggesting a negative effect of Rickettsia (16, 97

61). Reproductive manipulation and Rickettsia-associated parthenogenesis have been shown in 98

the endoparasitoid Neochrysocharis formosa (42), and in the parasitoid wasp Pnigalio soemius 99

(37), as has Rickettsia-associated male-killing in beetles (71, 69), and involvement in oogenesis 100

of booklice (72). 101

To unravel additional biological functions for Rickettsia in B. tabaci, including possible 102

transmission routes, the spatial subcellular localization of the bacterium in internal organs and 103

cells must be determined. Little is known about the specific subcellular localization of Rickettsia 104

within arthropod hosts. To hypothesize possible biological functions and interactions with the 105

insect host, fluorescence and electron microscopy were used here to show that Rickettsia 106

occupies specific, previously undescribed niches and organs within B. tabaci’s digestive, salivary 107

and reproductive organs and cells. The presence of the bacterium in these organs gives some clue 108

as to the specific undescribed interactions and possible transmission routes, suggests possible 109

horizontal transfer of the bacterium during mating, and confirms a recent report suggesting that 110

horizontal transmission of Rickettsia might be plant-mediated (14). 111

112

MATERIALS AND METHODS 113

Insects and rearing conditions. Rickettsia-free and Rickettsia-containing B-biotype B. tabaci 114

populations were reared on cotton seedlings (Gossypium hirsutum L. cv. Acala) maintained 115

inside insect-proof cages and growth rooms under standard conditions of 25 ± 2 ºC, 60% relative 116

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humidity and a 14 h light/10 h dark photoperiod. Identification of the B biotype was based on 117

microsatellite markers (23). The two strains of B. tabaci biotype B used in this study were 118

established by selection of isofemale strain as previously described (11). The original B biotype 119

population used to establish the isofemale strains was collected from Ayalon valley in Israel 120

(31° 52′ 3″ N, 34° 57′ 34″ E) in 1996 and cultured under laboratory conditions. This population 121

tested positive for Portiera and Hamiltonella. 122

DNA extraction and PCR amplification. To confirm the presence of Rickettsia in each adult B. 123

tabaci individual, 20 whiteflies were individually homogenized in lysis buffer as previously 124

described (31.). The lysate was then incubated at 65 ºC for 15 min followed by incubation at 95 125

ºC for 10 min. The samples were tested for the presence of Rickettsia by PCR using Rickettsia-126

specific primers for amplification of the 16S rDNA gene fragment: Rb-F 5’-127

GCTCAGAACGAACGCTATC-3’ and Rb-R 5’-GAAGGAAAGCATCTCT GC-3’ (38). The 128

reaction was carried out in a 20-µl volume containing 2 µl template DNA lysate, 20 pmol of each 129

primer, 10 mM dNTPs, 1X DreamTaq buffer and 1 U DreamTaq DNA Polymerase (Fermentas). 130

PCR-amplified products were visualized on a 1% agarose gel containing ethidium bromide. To 131

detect Rickettsia in specific organs using PCR, B. tabaci midguts, stylets, primary salivary 132

glands and hemolymph were dissected under binocular in 1X phosphate buffered saline (PBS), 133

washed and subjected to the above-described lysis-PCR amplification. The reaction contained 10 134

µl of template DNA lysate. 135

FISH. FISH was performed as previously described (38). Briefly, specimens were fixed 136

overnight in Carnoy’s fixative (chloroform:ethanol:glacial acetic acid, 6:3:1, v/v), then 137

decolorized in 6% H2O2 in ethanol for 2 h and hybridized overnight in hybridization buffer (20 138

mM Tris-HCl pH 8.0, 0.9 M NaCl, 0.01% w/v sodium dodecyl sulfate, 30% v/v formamide) 139

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containing 10 pmol fluorescent probes per ml. Dissected midguts, salivary glands and ovaries 140

were fixed for 5 min in Carnoy’s fixative and hybridized overnight. For specific targeting of 141

Portiera and Rickettsia, BTP1-Cy3 (5’-Cy3-TGTCAGTGTCAGCCCAGAAG-3’) and Rb1-Cy5 142

(5’-Cy5-TCCACGTCG CCGTCTTGC-3’) probes were used, respectively. Nuclei were stained 143

with 4’,6’-diamidino-2-phenylindole (DAPI) (0.1 mg ml-1). The stained samples were mounted 144

whole in hybridization buffer and viewed under an IX81 Olympus FluoView500 confocal 145

microscope. For each stage, at least 15 specimens were viewed under the microscope to confirm 146

reproducibility. Optical sections (0.7-1.0 μm thick) were prepared from each specimen. 147

Specificity of detection was confirmed using no-probe and Rickettsia-free whitefly controls. 148

Electron microscopy. Detached adult whitefly abdomens and heads were fixed overnight in 149

2.5% glutaraldehyde in 1X PBS at 4 ºC and processed using a standard method for TEM: rinsing 150

in 1X PBS buffer, osmification, another rinse in 1X PBS buffer, dehydration in an ascending 151

ethanol series, acetone incubation and embedding in epoxy resin Agar 100 (Agar Scientific, 152

Essex, England). Thin sections (60–90 nm) were cut using an ultramicrotome, stained with 153

aqueous uranyl acetate and lead citrate and examined in a Tecnai 12 electron microscope 154

(Philips/FEI, Eindhoven, The Netherlands). 155

156

RESULTS 157

General distribution of Rickettsia in the adult hemolymph. The distribution of Rickettsia was 158

monitored in whole insects and dissected organs by means of FISH and TEM. The bacterium 159

was generally distributed throughout the body cavity, appearing in the head, thorax and abdomen 160

(Fig. 1A). In some cases, the bacterium was heavily concentrated around bacteriocytes, and was 161

less observable in other locations (Figs. 1B–F). The latter phenotype was not significantly 162

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correlated with whitefly sex or age. Many serial confocal and TEM sections indicated that 163

Rickettsia is not present in bacteriocytes in the adult female stage, even when the bacterium cells 164

are highly abundant around bacteriocytes. In only one case Rickettsia-like cells were observed 165

attached to the outer surface of the bacteriocyte, and potentially internalized by the bacteriocyte 166

(Fig. 1F). These observations suggest that the mode of vertical transmission to the next 167

generation via bacteriocytes cannot explain the high abundance and perfect vertical transmission 168

of Rickettsia from the female to its offspring. 169

170 Oocyte-mediated vertical transmission of Rickettsia. We tested the hypothesis that Rickettsia 171

hitchhikes with the bacteriocyte for vertical transmission via the oocyte, as was previously 172

described for the other endosymbionts in whiteflies (12, 20, 39, 40, 65). The results indicated 173

that Rickettsia is the exception. The possibility for vertical transmission via free bacteriocytes 174

was excluded based on TEM and confocal serial sections (Fig. 1). Rickettsia was not seen 175

penetrating bacteriocytes that were already contained in the oocyte or free in the hemolymph, 176

suggesting that this is not a route of oocyte entry. The path of Rickettsia entrance into developing 177

oocytes was followed, focusing on different developmental stages of the oocyte. Indeed, 178

Rickettsia invaded the oocytes in the very early stages of development, and high levels of 179

bacterial cells were concentrated inside the oocytes at all developmental stages, especially the 180

younger stages 1 and 2 (Figs. 2A and C-F). The concentration of bacterial cells decreased in the 181

more mature oocytes, especially in their central part (Figs. 2A–E), while they were not observed 182

in free bacteriocyte cells surrounding the developing oocytes (Fig. 2F). The location of Rickettsia 183

in the developing oocytes was further explored using serial TEM sections. Young oocytes in 184

stages 1 to 3 contained larger amounts of Rickettsia cells in the cytoplasm and in the surrounding 185

follicular cells (Figs. 3A–C). However, oocytes in later stages 4 and 5 of development contained 186

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bacterial cells mostly in the surrounding follicular cells and less Rickettsia inside the oocyte 187

cytoplasm (Figs. 3D and E). At stage 5 of development, the oocyte is almost completely filled 188

with fat vacuoles and droplets and bacterial cells were not observed in the cytoplasm (Fig. 3E). 189

190 Organ- and tissue-specific infection. The general distribution of Rickettsia in B. tabaci as 191

observed by FISH led us to monitor its presence in other whitefly organs and subcellularly, using 192

both TEM and FISH analyses. The organs and tissues analyzed were the digestive and salivary 193

systems including the midgut, salivary glands and stylet. Furthermore, the male and female 194

reproductive organs were analyzed for infection by Rickettsia, and the possible sexual transfer of 195

the bacterium between infected and healthy individuals was tested. Other tissues that were tested 196

for infection were flight muscles in the thorax and fat bodies in the abdomen. 197

Experiments employing dissected midguts and Rickettsia-specific probe showed that the 198

midgut contains very high amounts of bacterial cells, unlike many of the other organs in B. 199

tabaci (Fig. 4). Rickettsia cells sometimes clustered in groups inside vacuolar-like structures 200

(Fig. 4A and Figs. 5C and D), and were easily distinguished by TEM and FISH (Fig. 4A and C 201

and Figs. 5A and B). The bacterium cells were never observed in the midgut lumen (Figs. 4B and 202

C and Figs. 5A and C). Midguts dissected from younger 1-2 days old males and females, as well 203

as older 7-14 days individuals contained similar localization patterns of Rickettsia. 204

FISH analysis detected Rickettsia in the primary salivary glands (Fig. 6B), and this result 205

was confirmed using TEM (Figs. 6C and D). The bacterial cells were located in cells 1 and 2 206

described by Ghanim et al. (36). These cells contain exceptionally large nuclei, electron-dense 207

granules and lipid-like accumulations, but their cytoplasm is less dense than that of the other 208

cells in the gland (36), and were hypothesized to act in producing the gelling saliva required for 209

the stylet penetration in the plant leaf. Using FISH, we were able to confirm the presence of 210

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Rickettsia in the whitefly stylet, along the interlocked maxillae through which the food and 211

salivary canals pass (Fig. 7). Interestingly, FISH signal was also observed on the mandibles that 212

surround these two canals (Fig. 7). At this resolution, we were not able to determine whether the 213

signal exists only in the food, salivary, or both canals. 214

The reproductive organs of both B. tabaci males and females were monitored for the 215

presence of Rickettsia. The bacterium was detected by FISH in dissected testes from B. tabaci 216

males (Figs. 8A–D) and dissected spermathecae from fertilized females (Figs. 8E and F). 217

Interestingly, the labeling was observed in all stages of spermatogenesis in the primary cells of 218

the primary glands in the testes and spermathecae; however, less Rickettsia cells were associated 219

with the mature spermatids (Fig. 8D). This observation suggested that Rickettsia might be 220

sexually transmitted between individuals, one mechanism for horizontal transfer, thus affording a 221

potential route for Rickettsia maintenance in B. tabaci populations. Indeed, copulation 222

experiments that we conducted on artificial diet between 30 Rickettsia-infected females with 30 223

uninfected males and vice versa, showed that Rickettsia could be detected by PCR in some of the 224

uninfected counterparts (data not shown). 225

Further tissues in which Rickettsia cells were detected are muscle band cells in the flight 226

muscles located in the thorax of adult males and females using TEM ultrathin sections (Fig. 9A), 227

and fat bodies in the hemolymph of both sexes (Figs. 9B and C ). 228

229

DISCUSSION 230

In the present study we obtained new data regarding the infection of B. tabaci by Rickettsia. We 231

further showed organ-specific distribution and possible relevance to the high abundance of 232

Rickettsia in the insect populations (45). The high abundance of Rickettsia in the hemolymph, 233

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especially around bacteriocytes, first suggested possible vertical transmission via the oocyte by 234

hitchhiking with bacteriocyte as was previously suggested (38). Although it is widely accepted 235

that many bacterial endosymbionts are vertically transmitted into newly developed oocytes with 236

bacteriocytes, especially in whiteflies (12, 20, 38, 39, 40, 65), the results presented here refute 237

this hypothesis and show that Rickettsia is the exception (Fig. 1). TEM serial sections did not 238

detect Rickettsia cells inside bacteriocytes, and only the abundant primary symbiont Portiera and 239

the secondary symbiont Hamiltonella, were always associated with bacteriocytes (Figs. 2 and 3). 240

TEM sections through the developing oocytes and bacteriocytes showed that younger oocytes are 241

heavily infected with Rickettsia cells, which are mostly excluded and concentrate in follicular 242

cells, with development. It is thus likely that Rickettsia cells are transferred to the next 243

generation via the egg by residing in the follicular cells. The observation of younger oocytes 244

being filled with bacterial cells corresponds with the fact that young oocytes are more permeable 245

to materials in the hemolymph than developed ones, which exhibit more selective barriers. 246

Follicular cells facilitate the uptake of various materials from the hemolymph to the oocyte and 247

are involved in the synthesis and transport of precursors of both the internal vitelline and external 248

chorion envelopes that surround the oocyte (53). Rickettsia has been shown to invade the 249

follicular cells of parasitic wasps of B. tabaci (18), but bacterial cells were not observed beyond 250

the follicular cells. Other bacterial symbionts such as Wolbachia in Drosophila melanogaster 251

(32), and Cardinium in the leafhopper Scaphoideus titanus (60), have been shown to invade the 252

reproductive system, including follicular cells, ensuring their own transmission to the next 253

generation. Our observation of Rickettsia invading the follicular cells and the cytoplasm of 254

developing oocytes suggests that this bacterium uses the oocyte, not the bacteriocyte route, to 255

ensure its transmission to the next generation. 256

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For the first time, we show that B. tabaci midgut is heavily infected with Rickettsia. It is 257

unclear how or when Rickettsia enters and becomes established in the midgut cells. One 258

possibility is the oocyte and egg cells that give rise to intestinal tissue in later developmental 259

stages. The high concentration of Rickettsia in the midgut and its specific localization in the 260

vacuoles (Figs. 5B and D), suggest a possible role in food digestion. Midgut-associated 261

symbionts have been reported from many arthropods, such as the Mediterranean fruit fly, tsetse 262

fly, silkworms, termites and stinkbugs, among many others (46). The diversity of midgut 263

microbes is enormous: they belong to various genera and exhibit different localization patterns, 264

modes of transmission and functional roles, depending on both the bacteria and the host (25, 29). 265

Intestinal microbes may contribute to food digestion, provide essential amino acids and vitamins 266

to the host, fix nitrogen and keep out potentially harmful microbes (2, 4, 5, 10, 66, 35). Gut 267

microorganisms also possess metabolic properties that are absent in insects, thus enabling the 268

phytophagous insects to overcome biochemical barriers to herbivory by detoxifying plant 269

allelochemicals such as flavonoids, tannins, and alkaloids (7, 25, 26). However, the gut bacteria 270

explored to date are phylogeneticlly distant from the α-proteobacterial group of Rickettsia and 271

nothing is known about the latter's functional role in the digestive system. 272

Interestingly, Rickettsia cells were found in the primary salivary glands (Fig. 6). 273

Occurrence of bacteria in the salivary glands has been reported from different insect species, and 274

has been suggested to serve as a route for its horizontal transmission to plants or other 275

intermediate hosts (28, 54, 59, 60). The first and strongest evidence of horizontal transmission of 276

symbiotic bacteria via an insect's plant host was reported for the leafhopper Euscelidius 277

variegates (59) and the pathogenic symbiont BEV. Similar to other sap-sucking arthropods, the 278

horizontal transmission of B. tabaci symbionts requires the passage of the bacterial cells through 279

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several barriers, most importantly from salivary gland cells through the salivary duct to the 280

stylet. Our results show that presence of Rickettsia in the whitefly stylet is restricted to the 281

interlocked maxillae through which the food and salivary canals pass (Fig. 7). Interestingly, 282

FISH signal was also observed on the mandibles that surround these two canals (Figure 7). At 283

this resolution, we were not able to determine whether the signal exists in the food, salivary, or 284

both canals; however, the presence of the bacterium in either one of these canals suggests its 285

passage into or out of the whitefly body with plant sap. It has been recently suggested that 286

Rickettsia is horizontally transmitted through the plant host (14), where it was observed in the 287

plant sieve elements following feeding of Rickettsia-infected whiteflies and was also acquired 288

from the plant by Rickettsia-free whiteflies. Combining our microscopy findings with the recent 289

report of Caspi-Fluger et al. (14) provides stronger evidence for the horizontal transfer of 290

Rickettsia in B. tabaci through the plant host. 291

Our results indicated the infection of male and female reproductive tissues by Rickettsia, 292

and preliminary copulation experiments that we performed, using artificial diet, suggest possible 293

transfer of Rickettsia during mating (Fig. 8 and unpublished data). These results, in addition with 294

the recent study suggesting horizontal transmission through the plant (14), might explain the 295

exceptionally high prevalence of Rickettsia in natural populations, such as its establishment and 296

near fixation in populations of B. tabaci in Arizona in just 6 years (45). In the later study, 297

Rickettsia-infected whiteflies produced more offspring, had higher survival rates, developed 298

faster and produced a higher proportion of daughters. The observed sex ratio bias might be 299

attributed in part to possible effects of Rickettsia on the Rickettsia-infected sperm in the female 300

spermatheca, which is used by the female to fertilize eggs. In this arrhenotokous mode of 301

reproduction, fertilized eggs develop to females while unfertilized eggs develop to males. 302

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Wolbachia is the most studied microorganism with respect to associations with insect 303

reproductive organs, which induce cytoplasmic incompatibility (CI) and reproductive 304

manipulations in several insect species, and was associated with breakdown of spermatogenesis 305

(30). Several other bacterial symbionts, which reside in male and female reproductive organs and 306

induce reproductive manipulations, were further described, including Cardinium (21, 73, 74), 307

Candidatus Blochmannia floridanus (62) and Arsenophonus (70). A spotted fever group 308

Rickettsia was previously described in the spermatogonia, spermatocytes and maturing 309

spermatids of its male tick vector Ixodes ricinus, and it could be sexually transmitted to females, 310

but it did not have negative effects on the developing eggs (44). 311

The presence of Rickettsia in muscle cells was somewhat surprising, and it is unclear 312

whether this has any effect on the muscle-related biology of the whitefly, such as flight. Several 313

bacterial symbionts that have been previously localized to thoracic and muscle tissues, of their 314

hosts were associated with inducing negative and even pathogenic effects on the host. The best 315

studied examples of Rickettsia species that infect muscle tissues stem from pathogenic Rickettsia 316

species and their interactions with their arthropod vectors. Few examples include the two major 317

groups of pathogenic Rickettsia: the spotted fever and typhus groups, and their interactions with 318

their tick vectors (64), and Anaplasma marginales (Rickettsiales: Anaplasmataceae) in the male 319

Rocky Mountain wood tick Dermacentor andersoni (48). 320

The association of Rickettsia with B. tabaci fat bodies was not expected as the presence 321

and functional association of endosymbionts in fat bodies is not common in insects. Only 322

endosymbionts from the Blattabacteria that co-evolved with termites and cockroaches were 323

reported from the fat body, but no specific functional role for these endosymbionts has been 324

assigned (19, 50, 52). In whiteflies, vitellogenin and possibly other important proteins are 325

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produced in the fat body, and thus the presence of Rickettsia might indicate a role in these 326

processes. 327

In conclusion, FISH and TEM analyses showed the presence of Rickettsia endosymbiont 328

in B. tabaci whiteflies. Rickettsia was localized to major organs involved in the host's 329

reproductive, feeding, circulation and secretion systems. Interestingly, the broad spectrum of 330

Rickettsia-infected tissues in B. tabaci documented in this study resembles that found for the 331

pathogenic Rickettsia felis in cat fleas (1, 9, 51). Further investigation is warranted to understand 332

the mechanism by which Rickettsia reaches and becomes established in these organs and its 333

possible roles there. 334

335

336

ACKNOWLEDGMENTS 337

We thank Eduard Belausov, Vered Holdengreber and Svetlana Kontsedalov for technical 338

assistance. This research was supported by Binational Science Foundation (BSF) grant 2007045, 339

Israel Science Foundation (ISF) grant 884/07. This is contribution number 501/12 from ARO 340

publications. 341

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552

553

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FIGURE LEGENDS 554

FIG 1 Rickettsia localization in Bemisia tabaci adults by TEM and FISH analysis with 555

Rickettsia-specific probe (blue) and a probe for Portiera (red). (A) Rickettsia occupies most of 556

the body cavity while Portiera is located inside bacteriocytes in the abdomen. (B) Rickettsia is 557

sometimes found at exceptionally high concentrations around the bacteriocytes. (C) Details of 558

the box in (b). (D) TEM image showing three bacteriocytes (B) in the hemolymph surrounded by 559

high concentrations of Rickettsia (R) cells. (E) Rickettsia (R) cells surrounding a bacteriocyte 560

that harbors Portiera (P) and Hamiltonella (H) in the hemolymph of B. tabaci biotype B. (F) Box 561

in E showing a bacteriocyte surrounded by Rickettsia (R) and some Rickettsia-like cells attached 562

to or inside the bacteriocyte. Bar in panel D, 2.5 μm; in panel E, 2 μm and in panel F, 0.5 μm. 563

N, nucleus. 564

565

FIG 2 FISH of Bemisia tabaci ovaries using Rickettsia-specific probe (green), specific probe for 566

Portiera (red) and DAPI staining of the nuclei (blue). (A) Stages 1–4 of ovary development 567

showing very high concentrations of Rickettsia cells invading younger ovaries. (B) One ovary at 568

stage 4 of development showing the distribution of Rickettsia cells in and around it. (C) A focal 569

plane showing the presence of Rickettia cells inside the ovary and at the center of the oocyte (O). 570

(D) Ovary at stage 5 of development showing the included Rickettsia-free bacteriocyte (B). (E) 571

Same as in D with only one focal plane showing Rickettsia cells inside the oocyte. (F) Dissected 572

ovaries and Rickettsia-free bacteriocytes (B) in the hemolymph of Bemisia tabaci. 573

574

FIG 3 TEM sections of Bemisia tabaci abdomen. (A) Stage 1 ovary filled with Rickettsia in 575

follicular cells (FC). (B) Stage 2 ovary filled with and surrounded by Rickettsia (R and arrows). 576

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(C) Stage 3 ovary with follicular cells (FC) and Rickettsia (arrows) mainly in the oocyte (O). (D) 577

Follicular cells (FC) filled with and surrounded by Rickettsia (R and arrows). (E) Mature egg 578

showing the oocyte (O) with Rickettsia excluded and only in the surrounding follicular cells 579

(arrows). H, hemolymph; FV, fat vacuole; FD, fat-dense granules. Bar in panel A, 4 μm; in 580

panel B, 2.5 μm; in panel C, 1 μm ; in panel D, 1 μm; in panel E, 2 μm. 581

582

FIG 4 FISH of dissected midgut using Rickettsia-specific probe (red) and DAPI staining. (A) 583

Whole midgut showing Rickettsia concentrated in clusters in some midgut cells (arrows). (B) 584

DAPI staining showing high concentrations of Rickettsia in the cytoplasm of midgut cells. (C) 585

Same as in B but with FISH using Rickettsia-specific probe showing that the cells stained with 586

DAPI are now labeled with the probe. C, ceca; FC, filter chamber; DM, descending midgut; AM, 587

ascending midgut; H, hindgut; L, lumen; N, nucleus. 588

589

FIG 5 TEM sections of Bemisia tabaci midgut. (A) Concentration of Rickettsia in midgut 590

epithelial cells (EC). (B) Details of the box in A. (C) Concentration of Rickettsia (R) in vacuoles 591

in midgut epithelial cells (EC). (D) Details of the box in C. L, lumen; N, nucleus. Bar in panel A, 592

3 μm; in panel B, 1 μm; in panel C, 2.5 μm ; in panel D, 1 μm. 593

594

FIG 6 TEM section and FISH using Rickettsia-specific probe (green) of Bemisia tabaci primary 595

salivary glands. (A) Two dissected primary salivary glands (PSG) stained with DAPI (blue). (B) 596

Same as in A, with Rickettsia-specific FISH. (C) TEM section in one primary salivary gland 597

(PSG) in the thorax, showing a giant cell and the nucleus (N). (D) Details of box in C showing 598

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Rickettsia-specific FISH in the gland and lipid-like accumulations (arrows). T, thorax; MB, 599

muscle band; R, Rickettsia. Bar in panel C, 2.5 μm; in panel D, 1 μm. 600

601

FIG 7 FISH of Bemisia tabaci stylet using Rickettsia-specific probe (red). Specific FISH signal 602

seen under bright field (A) and dark field (B) in the maxilla that form the food and salivary 603

canals (MX), and in the mandibles (MD) that were detached from the maxilla in this preparation. 604

LR, labrum. 605

606

FIG 8 FISH of Bemisia tabaci testes and spermathecae dissected from males and females, with 607

Rickettsia-specific probe (green) and DAPI staining (blue). (A) Two testicle (T) and two 608

accessory glands (AG) in the male genitalia dissected and stained with DAPI. The ducts 609

connecting the testicles to the common duct (CD) are also seen (arrows). (B) One testicle under 610

bright field showing the major locations of sperm production where the spermatogonia (sg), 611

spermatocytes (sy) and spermatids (st) or mature sperm are located. (C) The same testicle as in B 612

with Rickettsia-specific FISH (green) and DAPI staining. (D) The same testicle as in B and C 613

with Rickettsia-specific FISH only. (E) Spermatheca dissected from fertilized female showing 614

the sperm and Rickettsia-specific FISH (green). (F) Spermatheca seen in E with Rickettsia-615

specific FISH (green) only. 616

617

FIG 9 TEM section in Bemisia tabaci thorax and fat body in the hemolymph. (A) Rickettsia cells 618

(R) are located in one muscle cell close to the nucleus. (B) Several fat body cells (FB) filled with 619

fat vacuoles. (C) Details of the box in B showing Rickettsia cells in fat body cells (arrows). MB, 620

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muscle bands; N, nucleus; FV, fat vacuole. Bar in panel A, 1 μm; in panel B, 2.5 μm; in panel 621

C, 1 μm. 622

623

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