Abundance and Localization of Symbiotic Bacterial Communities in the Fly Parasitoid Spalangia cameroni.

Multicellular eukaryotes often host multiple microbial symbionts that may cooperate or compete for host resources, such as space and nutrients. Here, we studied the abundances and localization of four bacterial symbionts, Rickettsia, Wolbachia, Sodalis, and Arsenophonus, in the parasitic wasp Spalangia cameroni. Using quantitative PCR (qPCR), we measured the symbionts' titers in wasps that harbor different combinations of these symbionts. We found that the titer of each symbiont decreased as the number of symbiont species in the community increased. Symbionts' titers were higher in females than in males. Rickettsia was the most abundant symbiont in all the communities, followed by Sodalis and Wolbachia. The titers of these three symbionts were positively correlated in some of the colonies. Fluorescence in situ hybridization was in line with the qPCR results: Rickettsia, Wolbachia, and Sodalis were observed in high densities in multiple organs, including brain, muscles, gut, Malpighian tubules, fat body, ovaries, and testes, while Arsenophonus was localized to fewer organs and in lower densities. Sodalis and Arsenophonus were observed in ovarian follicle cells but not within oocytes or laid eggs. This study highlights the connection between symbionts' abundance and localization. We discuss the possible connections between our findings to symbiont transmission success. IMPORTANCE Many insects carry intracellular bacterial symbionts (bacteria that reside within the cells of the insect). When multiple symbiont species cohabit in a host, they may compete or cooperate for space, nutrients, and transmission, and the nature of such interactions would be reflected in the abundance of each symbiont species. Given the widespread occurrence of coinfections with maternally transmitted symbionts in insects, it is important to learn more about how they interact, where they are localized, and how these two aspects affect their co-occurrence within individual insects. Here, we studied the abundance and the localization of four symbionts, Rickettsia, Wolbachia, Sodalis, and Arsenophonus, that cohabit the parasitic wasp Spalangia cameroni. We found that symbionts' titers differed between symbiotic communities. These results were corroborated by microscopy, which shows differential localization patterns. We discuss the findings in the contexts of community ecology, possible symbiont-symbiont interactions, and host control mechanisms that may shape the symbiotic community structure.

Genomic characterization of viruses associated with the parasitoid Anagyrus vladimiri (Hymenoptera: Encyrtidae).

Knowledge on symbiotic microorganisms of insects has increased dramatically in recent years, yet relatively little data are available regarding non-pathogenic viruses. Here we studied the virome of the parasitoid wasp Anagyrus vladimiri Triapitsyn (Hymenoptera: Encyrtidae), a biocontrol agent of mealybugs. By high-throughput sequencing of viral nucleic acids, we revealed three novel viruses, belonging to the families Reoviridae [provisionally termed AnvRV (Anagyrus vladimiri reovirus)], Iflaviridae (AnvIFV) and Dicistroviridae (AnvDV). Phylogenetic analysis further classified AnvRV in the genus Idnoreovirus, and AnvDV in the genus Triatovirus. The genome of AnvRV comprises 10 distinct genomic segments ranging in length from 1.5 to 4.2 kb, but only two out of the 10 ORFs have a known function. AnvIFV and AnvDV each have one polypeptide ORF, which is typical of iflaviruses but very un-common among dicistroviruses. Five conserved domains were found along both the ORFs of those two viruses. AnvRV was found to be fixed in an A. vladimiri population that was obtained from a mass rearing facility, whereas its prevalence in field-collected A. vladimiri was ~15 %. Similarly, the prevalence of AnvIFV and AnvDV was much higher in the mass rearing population than in the field population. The presence of AnvDV was positively correlated with the presence of Wolbachia in the same individuals. Transmission electron micrographs of females' ovaries revealed clusters and viroplasms of reovirus-like particles in follicle cells, suggesting that AnvRV is vertically transmitted from mother to offspring. AnvRV was not detected in the mealybugs, supporting the assumption that this virus is truly associated with the wasps. The possible effects of these viruses on A. vladimiri's biology, and on biocontrol agents in general, are discussed. Our findings identify RNA viruses as potentially involved in the multitrophic system of mealybugs, their parasitoids and other members of the holobiont.

Wolbachia influence on the fitness of Anagyrus vladimiri (Hymenoptera: Encyrtidae), a bio-control agent of mealybugs.

BACKGROUND: Like numerous other animals, biocontrol agents (BCAs) of arthropod pests carry various microorganisms that may have diverse effects on the biology of their eukaryote hosts. We postulated that it is possible to improve the efficacy of BCAs by manipulating the composition of their associated microbiota. The parasitoid wasp Anagyrus vladimiri (Hymenoptera: Encyrtidae) from a mass-rearing facility was chosen for testing this hypothesis. RESULTS: High-throughput sequencing analysis indicated that fungal abundance in A. vladimiri was low and variable, whereas the bacterial community was dominated by the endosymbiont Wolbachia. Wolbachia was fixed in the mass-rearing population, whereas in field-collected A. vladimiri Wolbachia's prevalence was only approximately 20%. Identification of Wolbachia strains from the two populations by Multi Locus Sequence Typing, revealed two closely related but unique strains. A series of bioassays with the mass-rearing Wolbachia-fixed (W+ ) and a derived antibiotic-treated Wolbachia-free (W- ) lines revealed that: (i) Wolbachia does not induce reproductive manipulations; (ii) W- females have higher fecundity when reared individually, but not when reared with conspecifics; (iii) W+ females outcompete W- when they share hosts for oviposition; (iv) longevity and developmental time were similar in both lines. CONCLUSIONS: The findings suggest that W+ A. vladimiri have no clear fitness benefit under mass-rearing conditions and may be disadvantageous under lab-controlled conditions. In a broader view, the results suggest that augmentative biological control can benefit from manipulation of the microbiome of natural enemies.

Horizontal Transmission of Microbial Symbionts Within a Guild of Fly Parasitoids.

Many insects harbor facultative microbial symbionts which affect the ecology of their hosts in diverse ways. Most symbionts are transmitted vertically with high fidelity, whereas horizontal transmission occurs rarely. Parasitoid larvae feed on a single host and are in close physical contact with it, providing an ecological opportunity for symbionts' horizontal transmission, but there is little empirical evidence documenting this. Here we studied horizontal transmission of three bacterial symbionts-Rickettsia, Sodalis, and Wolbachia-between three fly pupal ectoparasitoid species: Spalangia cameroni, S. endius, and Muscidifurax raptor. Muscidifurax raptor readily parasitized and successfully developed on the Spalangia spp., while the inverse did not happen. The two Spalangia spp. attacked each other and conspecifics in very low rates. Symbiont horizontal transmissions followed by stable vertical transmission in the recipient species were achieved, in low percentages, only between conspecifics: Wolbachia from infected to uninfected M. raptor, Rickettsia in S. endius, and Sodalis in S. cameroni. Low frequency of horizontal transmissions occurred in the interspecific combinations, but none of them persisted in the recipient species beyond F4, at most. Our study is one of few to demonstrate symbionts' horizontal transmission between hosts within the same trophic level and guild and highlights the rarity of such events.

Effects, interactions, and localization of Rickettsia and Wolbachia in the house fly parasitoid, Spalangia endius.

Many insect species harbor facultative microbial symbionts that affect their biology in diverse ways. Here, we studied the effects, interactions, and localization of two bacterial symbionts-Wolbachia and Rickettsia-in the parasitoid Spalangia endius. We crossed between four S. endius colonies-Wolbachia only (W), Rickettsia only (R), both (WR), and none (aposymbiotic, APS) (16 possible crosses) and found that Wolbachia induces incomplete cytoplasmic incompatibility (CI), both when the males are W or WR. Rickettsia did not cause reproductive manipulations and did not rescue the Wolbachia-induced CI. However, when R females were crossed with W or WR males, significantly less offspring were produced compared with that of control crosses. In non-CI crosses, the presence of Wolbachia in males caused a significant reduction in offspring numbers. Females' developmental time was significantly prolonged in the R colony, with adults starting to emerge one day later than the other colonies. Other fitness parameters did not differ significantly between the colonies. Using fluorescence in situ hybridization microscopy in females, we found that Wolbachia is localized alongside Rickettsia inside oocytes, follicle cells, and nurse cells in the ovaries. However, Rickettsia is distributed also in muscle cells all over the body, in ganglia, and even in the brain.

Heat Adaptation of the House Fly (Diptera: Muscidae) and Its Associated Parasitoids in Israel.

Insects are ectothermic organisms; hence, all aspects of their biology are strongly influenced by ambient temperatures. Different insect species respond differently with phenotypic plasticity and/or genetic adaptation to changing temperatures. Here, we tested the thermal adaptation of the house fly and three of its parasitoids species by comparing life-history parameters in populations from a hot climate region (Jordan Valley) and from a moderate-climate region (Galilee). No significant differences were found between the two house fly populations, both under hot and moderate experimental conditions. Life-history parameters of the parasitoids (Muscidifurax raptor Girault & Sanders, Spalangia endius Walker, and Spalangia cameroni Perkins [Hymenoptera: Pteromalidae]) varied markedly between origins, species, sexes, and experimental conditions. Of the three species tested, only M. raptor collected in the Jordan Valley proved better adapted to experimental heat conditions, compared to its counterpart population that was collected in the Galilee. Additionally, we tested the effect of elevating temperatures on a house fly lab population for 17 consecutive generations and found no evidence for heat adaptation. We discuss our results in the context of house fly control and global warming.

Effect of Fluctuating High Temperatures on House Flies (Diptera: Muscidae) and Their Principal Parasitoids (Muscidifurax spp. and Spalangia spp. [Hymenoptera: Pteromalidae]) From the United States.

Colonies of house flies (Musca domestica L. [Diptera: Muscidae]) and four species of parasitoids (Muscidifurax raptor Girault and Sanders, Muscidifurax zaraptor Kogan and Legner, Spalangia cameroni Perkins and Spalangia endius Walker) were established by making collections from dairy farms near Bell, FL, Beatrice, NE, Minneapolis, MN, and San Jacinto, CA. Colonies were assessed for heat tolerance by comparing life history parameters at 25-27°C and fluctuating hot (26.7-41.7°C) temperatures. Muscidifurax raptor, S. cameroni, and S. endius produced 24-28% as many progeny under hot conditions as at 25°C. Colonies of M. zaraptor were more heat-tolerant and produced an average 46.9% as many progeny under the hot regime compared with moderate conditions. There was little evidence for higher heat tolerance in parasitoid populations from historically hot locations (CA desert and FL). Colonies of M. raptor and S. endius that had been in culture for 24 yr were the least heat-tolerant with regard to progeny production. House flies collected from the same locations varied little in longevity, fecundity, or egg-to-adult survival under either hot or moderate regimes. Flies reared under hot conditions laid about half as many eggs (89/female) and had about half the egg-adult survival rate (47.3%) under hot compared with moderate conditions, indicating that heat stress had less effect on flies than on all of the parasitoids except M. zaraptor. An attempt to select for heat tolerance in flies by subjecting them to incremental increases in rearing temperatures for 20 generations resulted in little change in tolerance among the selected flies.

Identification and characterization of bacterial symbionts in three species of filth fly parasitoids.

Facultative bacterial symbionts are widespread among insects and have diverse effects on their biology. Here, we focused on bacterial symbionts of three ecologically and economically important filth flies parasitoid species-Spalangia cameroni, Spalangia endius and Muscidifurax raptor. Both Spalangia species harbored a Sodalis bacterium that is closely related to Sodalis praecaptivus (a free-living bacterium) and to Sodalis symbionts of weevils. This is the only case of Sodalis infection in the important order Hymenoptera. We also found, for the first time in this parasitoid guild, a Rickettsia infecting the two Spalangia spp., albeit in much higher prevalence in S. cameroni. Molecular and phylogenetic analyses revealed that it is closely related to Rickettsia felis and other Rickettsia species from the 'transitional' group. All three parasitoid species harbored Wolbachia. Using multi-locus sequence typing, we found that M. raptor harbors a single Wolbachia strain whereas the Spalangia spp. have multiple strains. By controlled crossings, we found that Wolbachia infection in S. endius causes incomplete cytoplasmic incompatibility and increased longevity, thereby promoting Wolbachia's spread. In contrast, no effects of Wolbachia on the reproduction and longevity of M. raptor were found. This study underscores the diversity and nature of symbiotic interactions between microbes and insects.

An exceptional family: Ophiocordyceps-allied fungus dominates the microbiome of soft scale insects (Hemiptera: Sternorrhyncha: Coccidae).

Hemipteran insects of the suborder Sternorrhyncha are plant sap feeders, where each family is obligately associated with a specific bacterial endosymbiont that produces essential nutrients lacking in the sap. Coccidae (soft scale insects) is the only major sternorrhynchan family in which obligate symbiont(s) have not been identified. We studied the microbiota in seven species from this family from Israel, Spain and Cyprus, by high-throughput sequencing of ribosomal genes, and found that no specific bacterium was prevalent and abundant in all the tested species. In contrast, an Ophiocordyceps-allied fungus sp.-a lineage widely known as entomopathogenic-was highly prevalent. All individuals of all the tested species carried this fungus. Phylogenetic analyses showed that the Ophiocordyceps-allied fungus from the coccids is closely related to fungi described from other hemipterans, and they appear to be monophyletic, although the phylogenies of the Ophiocordyceps-allied fungi and their hosts do not appear to be congruent. Microscopic observations show that the fungal cells are lemon-shaped, are distributed throughout the host's body and are present in the eggs, suggesting vertical transmission. Taken together, the results suggest that the Ophiocordyceps-allied fungus may be a primary symbiont of Coccidae-a major evolutionary shift from bacteria to fungi in the Sternorrhyncha, and an important example of fungal evolutionary lifestyle switch.

Effects of pyriproxyfen on wild populations of the housefly, Musca domestica, and compatibility with its principal parasitoids.

BACKGROUND: The housefly, Musca domestica L., is an important pest of animal agriculture. Effective fly management requires integration of manure management, mass trapping, biological control, and selective insecticide use. Insecticidal control of houseflies is difficult due to the rapidity of resistance development, yet the insect growth regulator pyriproxyfen (PPF) is one of few insecticides that may still be effective. Here, we tested the susceptibility of wild housefly populations in the USA and in Israel to PPF, as well as the effect of PPF on housefly parasitoids of the genera Muscidifurax and Spalangia. RESULTS: Most housefly populations from both countries were completely eliminated at PPF concentrations of 100 mg kg-1 (USA) and 600 mg kg-1 (Israel). One population from each country exhibited initial levels of PPF tolerance. PPF efficacy significantly decreased in cow manure. Emergence rates of parasitoids developing in PPF-treated hosts at concentrations of ≥600 mg kg-1 were significantly affected, whereas other fitness parameters were moderately to non-affected. CONCLUSIONS: PPF is still an effective tool for housefly control, but resistance management practices should be employed to avoid resistance. PPF is compatible with principal housefly parasitoids at concentrations <600 mg kg-1 , and is suitable for use in integrated pest management. © 2017 Society of Chemical Industry.

Diversity and Abundance of House Fly Pupal Parasitoids in Israel, with First Records of Two Spalangia Species.

Filth flies (Diptera: Muscidae), particularly the house fly, Musca domestica L., are global pests of livestock production. In this study, we characterized the fauna of house fly pupal parasitoids in Israel and identified factors affecting their diversity and abundance. The study, which included one round of sampling during the fall of 2013 and another round of sampling in the spring of 2014, encompassed 26 locations of common fly-breeding habitats: dairy, egg-laying, and goat farms throughout Israel. Nine parasitoid species were found: Spalangia cameroni Perkins, Spalangia endius Walker, Spalangia drosophilae Ashmead, Spalangia gemina Boucek, Spalangia nigroaenea Curtis, Pachycrepoideus vindemmiae Rondani, Muscidifurax raptor Girault and Sanders, Muscidifurax zaraptor Kogan & Legner (all Hymenoptera: Pteromalidae), and Dirhinus giffardii Silvestri (Hymenoptera: Chalcididae). This is the first record of S. gemina from the Palearctic, as well as the first record of S. drosophilae from the Middle East. The composition and relative abundance of parasitoid species varied markedly among localities, climatic regions (Mediterranean vs. desert), habitat types (dairy vs. egg-laying vs. goat farm), and seasons. Overall, parasitoid richness in egg-laying farms was two- and sevenfold higher than in dairy and goat farms, respectively, and three times higher in Mediterranean than desert climate. The significance and implications of our results for inundative biological control programs of filth flies are discussed.

Do Bacterial Symbionts Govern Aphid's Dropping Behavior?

Defensive symbiosis is amongst nature's most important interactions shaping the ecology and evolution of all partners involved. The pea aphid, Acyrthosiphon pisum Harris (Hemiptera: Aphididae), harbors one obligatory bacterial symbiont and up to seven different facultative symbionts, some of which are known to protect the aphid from pathogens, natural enemies, and other mortality factors. Pea aphids typically drop off the plant when a mammalian herbivore approaches it to avoid incidental predation. Here, we examined whether bacterial symbionts govern the pea aphid dropping behavior by comparing the bacterial fauna in dropping and nondropping aphids of two A. pisum populations, using two molecular techniques: high-throughput profiling of community structure using 16 S reads sequenced on the Illumina platform, and diagnostic polymerase chain reaction (PCR). We found that in addition to the obligatory symbiont, Buchnera aphidicola, the tested colonies of A. pisum harbored the facultative symbionts Serratia symbiotica, Regiella insecticola and Rickettsia, with no significant differences in infection proportions between dropping and nondropping aphids. While S. symbiotica was detected by both techniques, R. insecticola and Rickettsia could be detected only by diagnostic PCR. We therefore conclude that A. pisum's dropping behavior is not affected by its bacterial symbionts and is possibly affected by other factors.

Characteristics, phenotype, and transmission of Wolbachia in the sweet potato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), and its parasitoid Eretmocerus sp. nr. emiratus (Hymenoptera: Aphelinidae).

Wolbachia is a common intracellular bacterial endosymbiont of insects, causing a variety of effects including reproductive manipulations such as cytoplasmic incompatibility (CI). In this study, we characterized Wolbachia in the whitefly Bemisia tabaci and in the whitefly parasitoid Eretmocerus sp. nr. emiratus. We also tested for horizontal transmission of Wolbachia between and within trophic levels, and we determined the phenotype of Wolbachia in E. sp. nr. emiratus. Using multilocus sequence typing and phylogenetic analyses, we found that B. tabaci and E. sp. nr. emiratus each harbor a different and unique strain of Wolbachia. Both strains belong to the phylogenetic supergroup B. No evidence for horizontal transmission of Wolbachia between and within trophic levels was found in our study system. Finally, crossing results were consistent with a CI phenotype; when Wolbachia-infected E. sp. nr. emiratus males mate with uninfected females, wasp progeny survival dropped significantly, and the number of females was halved. This is the first description of CI caused by Wolbachia in the economically important genus Eretmocerus. Our study underscores the expectation that horizontal transmission events occur rarely in the dynamics of secondary symbionts such as Wolbachia, and highlights the importance of understanding the effects of symbionts on the biology of natural enemies.

Rapid spread of a bacterial symbiont in an invasive whitefly is driven by fitness benefits and female bias.

Maternally inherited bacterial symbionts of arthropods are common, yet symbiont invasions of host populations have rarely been observed. Here, we show that Rickettsia sp. nr. bellii swept into a population of an invasive agricultural pest, the sweet potato whitefly, Bemisia tabaci, in just 6 years. Compared with uninfected whiteflies, Rickettsia-infected whiteflies produced more offspring, had higher survival to adulthood, developed faster, and produced a higher proportion of daughters. The symbiont thus functions as both mutualist and reproductive manipulator. The observed increased performance and sex-ratio bias of infected whiteflies are sufficient to explain the spread of Rickettsia across the southwestern United States. Symbiont invasions such as this represent a sudden evolutionary shift for the host, with potentially large impacts on its ecology and invasiveness.

Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex.

Bemisia tabaci, an invasive pest that causes crop damage worldwide, is a highly differentiated species complex, divided into biotypes that have mainly been defined based on mitochondrial DNA sequences. Although endosymbionts can potentially induce population differentiation, specialization and indirect selection on mtDNA, studies have largely ignored these influential passengers in B. tabaci, despite as many as seven bacterial endosymbionts have been identified. Here, we investigate the composition of the whole bacterial community in worldwide populations of B. tabaci, together with host genetic differentiation, focusing on the invasive B and Q biotypes. Among 653 individuals studied, more than 95% of them harbour at least one secondary endosymbiont, and multiple infections are very common. In addition, sequence analyses reveal a very high diversity of facultative endosymbionts in B. tabaci, with some bacterial genus being represented by more than one strain. In the B and Q biotypes, nine different strains of bacteria have been identified. The mtDNA-based phylogeny of B. tabaci also reveals a very high nucleotide diversity that partitions the two ITS clades (B and Q) into six CO1 genetic groups. Each genetic group is in linkage disequilibrium with a specific combination of endosymbionts. All together, our results demonstrate the rapid dynamics of the bacterial endosymbiont-host associations at a small evolutionary scale, questioning the role of endosymbiotic communities in the evolution of the Bemisia tabaci species complex and strengthening the need to develop a metacommunity theory of inherited endosymbionts.

Almost there: transmission routes of bacterial symbionts between trophic levels.

Many intracellular microbial symbionts of arthropods are strictly vertically transmitted and manipulate their host's reproduction in ways that enhance their own transmission. Rare horizontal transmission events are nonetheless necessary for symbiont spread to novel host lineages. Horizontal transmission has been mostly inferred from phylogenetic studies but the mechanisms of spread are still largely a mystery. Here, we investigated transmission of two distantly related bacterial symbionts--Rickettsia and Hamiltonella--from their host, the sweet potato whitefly, Bemisia tabaci, to three species of whitefly parasitoids: Eretmocerus emiratus, Eretmocerus eremicus and Encarsia pergandiella. We also examined the potential for vertical transmission of these whitefly symbionts between parasitoid generations. Using florescence in situ hybridization (FISH) and transmission electron microscopy we found that Rickettsia invades Eretmocerus larvae during development in a Rickettsia-infected host, persists in adults and in females, reaches the ovaries. However, Rickettsia does not appear to penetrate the oocytes, but instead is localized in the follicular epithelial cells only. Consequently, Rickettsia is not vertically transmitted in Eretmocerus wasps, a result supported by diagnostic polymerase chain reaction (PCR). In contrast, Rickettsia proved to be merely transient in the digestive tract of Encarsia and was excreted with the meconia before wasp pupation. Adults of all three parasitoid species frequently acquired Rickettsia via contact with infected whiteflies, most likely by feeding on the host hemolymph (host feeding), but the rate of infection declined sharply within a few days of wasps being removed from infected whiteflies. In contrast with Rickettsia, Hamiltonella did not establish in any of the parasitoids tested, and none of the parasitoids acquired Hamiltonella by host feeding. This study demonstrates potential routes and barriers to horizontal transmission of symbionts across trophic levels. The possible mechanisms that lead to the differences in transmission of species of symbionts among species of hosts are discussed.

The presence of Rickettsia is associated with increased susceptibility of Bemisia tabaci (Homoptera: Aleyrodidae) to insecticides.

BACKGROUND: The presence of certain symbiotic microorganisms may be associated with insecticide resistance in insects. The authors compared the susceptibility of two isofemale lines, Rickettsia-plus and Rickettsia-free, of the sweet potato whitefly Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) to major insecticides from different chemical groups, including imidacloprid, acetamiprid, thiamethoxam, pyriproxyfen, spiromesifen and diafenthiuron. RESULTS: While the Rickettsia-plus and Rickettsia-free lines showed no differences in their susceptibility to imidacloprid and diafenthiuron, higher susceptibility of the Rickettsia-plus line to acetamiprid, thiamethoxam, spiromesifen and especially pyriproxyfen was observed. LC(90) values indicated that the Rickettsia-free line was 15-fold more resistant to pyriproxyfen than the Rickettsia-plus line. CONCLUSION: Findings indicate that the infection status of B. tabaci populations by Rickettsia is an important consideration that should be taken into account when performing resistance monitoring studies, and may help in understanding the dynamics of B. tabaci resistance, symbiont-pest associations in agricultural systems and the biological impact of Rickettsia on whitefly biology.

Identification and localization of a Rickettsia sp. in Bemisia tabaci (Homoptera: Aleyrodidae).

Whiteflies (Homoptera: Aleyrodidae) are sap-sucking insects that harbor "Candidatus Portiera aleyrodidarum," an obligatory symbiotic bacterium which is housed in a special organ called the bacteriome. These insects are also home for a diverse facultative microbial community which may include Hamiltonella, Arsenophonus, Fritchea, Wolbachia, and Cardinium spp. In this study, the bacteria associated with a B biotype of the sweet potato whitefly Bemisia tabaci were characterized using molecular fingerprinting techniques, and a Rickettsia sp. was detected for the first time in this insect family. Rickettsia sp. distribution, transmission and localization were studied using PCR and fluorescence in situ hybridizations (FISH). Rickettsia was found in all 20 Israeli B. tabaci populations screened but not in all individuals within each population. A FISH analysis of B. tabaci eggs, nymphs, and adults revealed a unique concentration of Rickettsia around the gut and follicle cells, as well as a random distribution in the hemolymph. We postulate that the Rickettsia enters the oocyte together with the bacteriocytes, leaves these symbiont-housing cells when the egg is laid, multiplies and spreads throughout the egg during embryogenesis and, subsequently, disperses throughout the body of the hatching nymph, excluding the bacteriomes. Although the role Rickettsia plays in the biology of the whitefly is currently unknown, the vertical transmission on the one hand and the partial within-population infection on the other suggest a phenotype that is advantageous under certain conditions but may be deleterious enough to prevent fixation under others.