Vitellogenin Facilitates Associations between the Whitefly and a Bacteriocyte Symbiont

ABSTRACT Integration between animal reproduction and symbiont inheritance is fundamental in symbiosis biology, but the underlying molecular mechanisms are largely unknown. Vitellogenin (Vg) is critical for oogenesis, and it is also a pathogen pattern recognition molecule in some animals. Previous studies have shown that Vg is involved in the regulation of symbiont abundance and transmission. However, the mechanisms by which an insect and its symbiont contribute to the function of Vg and how Vg impacts the persistence of insect-microbe symbiosis remain largely unclear. Symbionts are transovarially transmitted via maternal inheritance of the bacteriocytes in the whitefly Bemisia tabaci. Surprisingly, Vg is localized in bacteriocytes of whiteflies. Vg could be synthesized in whitefly bacteriocytes by the gene Vg expressed in these cells or exported into bacteriocytes from hemolymph via the Vg receptor. We further found that the juvenile hormone and “Candidatus Portiera aleyrodidarum” (here termed Portiera) control the level and localization of Vg in whiteflies. Immunocapture PCR revealed interactions between Vg and Portiera. Suppressing Vg expression reduced Portiera abundance as well as whitefly oogenesis and fecundity. Thus, we reveal that Vg facilitated the persistence of whitefly-bacteriocyte symbiont associations. This study will provide insight into the key role of Vg in the coevolution of insect reproduction and symbiont inheritance.

Vitellogenin (Vg) is a large glycolipophosphoprotein and a precursor of the major egg yolk protein vitellin in many oviparous animals (18). The proteins are transported to the ovaries and incorporated into developing oocytes via Vg receptor (VgR)-mediated endocytosis (18). Vg is critical for oogenesis because it provides essential nutrients such as amino acids, lipids, and vitamins (18)(19)(20). A common form of vertical inheritance is transovarial transmission, which is typical for hereditary symbionts (3,6,(21)(22)(23)(24). Some symbionts use the host's molecular and cellular machinery for targeting oocytes (23,24). For example, Spiroplasma in Drosophila interacts with Vg and migrates to oocytes by coopting the VgR-mediated endocytotic mechanism (24). Vg is also a pathogen pattern recognition molecule in some animals. Vg is capable of killing or binding bacteria via interaction with lipopolysaccharide and lipoteichoic acid, as in fish, or by binding to peptidoglycan, lipopolysaccharide, and zymosan, as in the honey bee Apis mellifera (25,26). Vg acts as an antimicrobial agent in the Asian honey bee, Apis cerana (27), and it is required for transgenerational immunity in A. mellifera (26). Vg expression influences the levels of Rickettsia in whiteflies and Wolbachia in planthoppers (28,29). Vg appears to play a key role in insect-symbiont interactions. However, the mechanisms by which insect symbionts regulate the function of Vg and how Vg impacts the persistence of insect-microbe symbiosis remain largely unclear.
Whitefly symbiosis is a useful model system to study the persistence of insect-microbe symbiosis. The whitefly Bemisia tabaci is a complex of more than 40 cryptic species (30), among which B. tabaci MEAM1 is an invasive and globally important agricultural pest (31). All B. tabaci species harbor the obligate symbiont "Candidatus Portiera aleyrodidarum" (here termed Portiera) in specialized insect cells called bacteriocytes, and they harbor up to four facultative symbiont lineages (32,33). In China, B. tabaci MEAM1 bears Portiera and "Candidatus Hamiltonella defensa" (here termed Hamiltonella) in the same bacteriocyte (34,35). Portiera and Hamiltonella are transovarially transmitted via maternal inheritance of the bacteriocytes (21,36). Portiera is involved in the synthesis of 10 essential amino acids (EAAs) (37,38), which may influence protein synthesis. Additionally, the juvenile hormone (JH) regulates the Vg level in many insects (39)(40)(41). Thus, we proposed that JH and Portiera may regulate the Vg level in whiteflies and that Vg could impact whitefly reproduction and symbiont persistence. To test this hypothesis, we examined the localization of Vg in both ovarioles and bacteriocytes of whiteflies, studied how whitefly JH and Portiera affect the level and localization of Vg, and investigated how Vg impacts the whitefly and bacteriocyte symbiont associations. We found that Vg is localized in both ovarioles and bacteriocytes of whiteflies, that whitefly JH and Portiera positively adjust the level and localization Vg in whiteflies, and that Vg affects whitefly reproduction and the abundance and transmission of bacteriocyte symbionts.

RESULTS
Vg is localized in bacteriocytes of whiteflies. We examined the subcellular location of the whitefly Vg protein in ovaries by using a previously produced monoclonal antibody against Vg protein (42). Immunofluorescence microscopy showed that Vg was expressed in whitefly ovarioles at vitellogenesis phase III and their associated bacteriocytes (Fig. 1A). Vg was located mainly in the follicle cells, nurse cells, and oocytes of ovarioles at vitellogenesis phase II (Fig. 1A) as well as the cytoplasmic regions near the cell surfaces in contact with the external medium of bacteriocytes (Fig. 1A). The absence of a signal in the negative controls without primary antibody confirmed the specificity of Vg in whitefly bacteriocytes (Fig. 1A).
A parallel immunoelectron microscopy assay confirmed that Vg was present within the cytoplasm of Portiera ( Fig. 1B and C) and on the peripheral regions of Portiera ( Fig. 1B and  D), which could be the cell membrane of Portiera or the insect symbiosome membrane in the bacteriocytes. There were more Vg gold dots within the cytoplasm of Portiera than on the peripheral regions of Portiera ( Fig. 1E; P = 0.042). Vg was absent within the cytoplasm or on the peripheral regions of Portiera ( Fig. 1F and G) when bacteriocyte sections were treated without primary antibody or gold-conjugated goat-anti-mouse IgG as the controls.
Vg is synthesized mainly in the fat body, moves through the hemolymph, and is transported from the hemolymph to oocytes via VgR present on the surface of oocytes (43,44). The bacteriocyte is vertically transmitted by adult female whiteflies, which have ovarioles, but not by adult males, which have no ovarioles (45). Immunofluorescence microscopy showed that Vg was highly expressed in bacteriocytes of female whiteflies compared to that in male whiteflies ( Fig. 1H and I). At this point, it is not clear whether Vg protein is inherently expressed or simply exported and present in the bacteriocytes. We further detected the expression of Vg and VgR in the whole body and bacteriocytes of female and male whiteflies. The expression level of Vg or VgR is similar in the whole body of female and male whiteflies at day 1 after emergence (Fig. 1J). In contrast, the expression level of Vg or VgR is significantly higher in bacteriocytes of female whiteflies than in male whiteflies at day 1 after emergence (Fig. 1K). The expression level of Vg is significantly higher, but the expression level of VgR is significantly lower, in bacteriocytes than in the whole body of female whiteflies (Fig. 1L). The expression level of Vg or VgR is significantly lower in bacteriocytes than in the whole body of male whiteflies (Fig. 1M). These results suggest that Vg in whitefly bacteriocytes is produced either by the gene Vg expressed in whitefly bacteriocytes or by protein Vg exportation from the hemolymph via VgR in both females and males.
JH controls Vg expression and localization in whiteflies. JH regulates the Vg level in many insects (39)(40)(41). To test whether JH impacts Vg expression and Vg localization, whiteflies were injected with the JH analog pyriproxyfen. Expression of Vg was significantly upregulated at day 2 after injection in the whole body of pyriproxyfen-injected wild-type whiteflies compared to that in control whiteflies ( Fig. 2A; P = 0.24 and 0.044 for day 1 and day 2, respectively). JH acid methyltransferase (JHAMT) is a key enzyme that converts JH acids or inactive precursors of JHs to active JHs during the final step of the JH synthesis pathway in insects (46). To test the effect of JH on Vg expression and Vg localization, whitefly JHAMT was silenced. JHAMT expression was reduced by 57% and 39% at 5 and 6 days, respectively, after microinjection with double-stranded RNAs (dsRNAs) ( Fig. 2B; P = 0.0079 and 0.0075). Expression of Vg was significantly downregulated in dsJHAMT (dsRNA specific to whitefly JHAMT)-injected whiteflies compared to that in dsGFP (dsRNA specific to GFP)injected whiteflies ( Fig. 2C; P = 0.023) at day 5 postinjection. To test whether silencing JHAMT influenced expression of Vg, we examined the subcellular location of the whitefly Vg protein in ovaries and bacteriocytes by using the monoclonal antibody against Vg protein (42). Immunofluorescence microscopy showed that Vg localization declined in both ovarioles and bacteriocytes of ovarioles at vitellogenesis phase III as well as in bacteriocytes of dsJHAMT-injected whiteflies compared to that in dsGFP-injected whiteflies at day 5 after microinjection ( Fig. 2D and E and see Fig. S1 in the supplemental material; P = 0.0033 and P = 0.011).
Portiera elimination represses whitefly oogenesis and Vg localization. Portiera elimination can reduce whitefly fecundity (14). Thus, the total number of ovarioles and Vg localization in ovarioles and bacteriocytes were examined in Portiera-infected F1 B. tabaci (1PBt) and Portiera-cured F1 B. tabaci (2PBt) female whiteflies. The Portiera abundance was reduced by 99.9%, and the abundance of Hamiltonella and Rickettsia was reduced by 99.8% and 91.9%, respectively, by rifampin treatment ( Fig. 3A; P , 0.0001). The total number of ovarioles was decreased significantly in 2PBt whiteflies compared to that in 1PBt whiteflies Repressing Vg reduces symbiont abundance and whitefly reproduction. Vg is indispensable for insect reproduction and also functions as a pathogen pattern recognition molecule in some animals (18,25,26). To investigate the effect of repressing Vg on the abundance of the two symbionts in bacteriocytes and on whitefly reproduction, whitefly Vg was silenced by microinjection with dsVg (dsRNA specific to whitefly Vg). Vg localization in ovarioles and bacteriocytes, the abundance of the two symbionts, and the numbers of ovarioles and eggs were then examined. Expression of whitefly Vg was reduced by 58%, 72%, and 34% at 1, 3, and 5 days, respectively, after dsVg injection ( Fig. 4A; P , 0.05). Western blot analysis showed that the Vg level was significantly decreased in dsVg-injected whiteflies compared with that in dsGFP-injected whiteflies ( Fig. 4B; P = 0.0013). As a result of the gene silencing, Vg localization was noticeably reduced in ovarioles and bacteriocytes at days 1, 3, and 5 after RNA interference (RNAi) treatment (

DISCUSSION
The integration of insect reproduction and symbiont inheritance is critical for the persistence of insect-microbe symbiosis. However, the molecular mechanisms underlying the integration and coevolution of animal reproduction and symbiont inheritance are largely unknown. In this study, we found that JH and Portiera upregulate the level and localization of Vg in whitefly ovaries and bacteriocytes, and in turn, Vg facilitates the abundance and transmission of Portiera and whitefly oogenesis and fecundity (Fig. 5). This study reveals that the multifunctional protein Vg has become the central player in the integration between symbiont inheritance and insect reproduction or development. Our data also indicate that the bacteriocyte's function with Vg expression is the key for whitefly symbiosis evolution. Although this interaction of Vg and symbionts should still be treated with caution to be considered universal, our results provide insight into the coevolution of insect reproduction and symbiont inheritance.
Generally, Vg is synthesized mainly in the fat body and transported to growing oocytes by membrane-bound VgR through receptor-mediated endocytosis (43,44). Surprisingly, we found that Vg is also present in whitefly bacteriocytes. Vg in whitefly bacteriocytes can be generated by the gene Vg expressed in whitefly bacteriocytes and exported from the hemolymph via VgR expressed in whitefly bacteriocytes. The expression of the genes Vg and VgR as well as the protein Vg in whitefly bacteriocytes is unique in insect symbiosis. JH can control the Vg level in whiteflies as in most hemipterans by regulating Vg gene transcription (40,41). In contrast, Portiera determines the level and localization of Vg in female adult whiteflies by directly influencing protein synthesis, as this symbiont can provide multiple EAAs. Portiera elimination decreases whitefly fecundity (13). Our previous work demonstrated that Portiera can cooperate with horizontally transferred genes of whiteflies for the Role of Vitellogenin in Whitefly Symbiosis mBio synthesis of lysine, which impacts whitefly fecundity (47). Vg is a precursor of the major egg yolk protein (18). Thus, Portiera can affect Vg synthesis in female adult whiteflies by providing EAAs, thereby influencing whitefly fecundity. Other bacteriocyte symbionts can also synthesize pantothenate or EAAs (2,48,49), thus influencing Vg levels in insects, too. Therefore, it could be common that host symbiosis impacts the Vg level in insects harboring bacteriocyte-associated symbionts.

Role of Vitellogenin in Whitefly Symbiosis mBio
Vg can provide critical nutrients for oogenesis and embryogenesis of insects, and it is also a pathogen pattern recognition molecule in some animals. Rice stripe virus and Tomato yellow leaf curl virus can bind to Vg for transovarial transmission in small brown planthoppers and whiteflies, respectively (50,51). The symbiont Spiroplasma uses the host Vg transovarial transportation system for vertical transmission in Drosophila (24), and the symbiont Nasuia interacts with Vg during vertical transmission in leafhoppers (52). We found that Vg was present within the cytoplasm and on the peripheral regions of Portiera. Repressing Vg reduced the abundance of Portiera in bacteriocytes. IC-PCR revealed that there are interactions between Vg and Portiera. Overall, the presence of Vg in the cytoplasm of the symbiont and the regulation of symbiont abundance by Vg indicate that Vg may have the function of the pathogen pattern recognition molecule as reported in fish and honeybees (53)(54)(55). The novel function of Vg facilitates the interaction between Vg and symbiont.
Although Portiera and Hamiltonella colocalize in the same bacteriocyte, our study showed that disruption of Vg negatively impacted Portiera localization in bacteriocytes but had no effect on Hamiltonella. That could be caused by the localization of Vg and symbionts in the bacteriocyte and the differential cell structure of Portiera and Hamiltonella. Vg was located mainly in the cytoplasmic regions near the cell surfaces in contact with the external medium of bacteriocytes (Fig. 1A, H, and I, 2E, 3D, and 4D to F). Portiera occupied the cytoplasmic regions of bacteriocytes, while Hamiltonella was distributed mainly around bacteriocyte nuclei (34,36,45). Thus, the localization of Vg in the bacteriocyte is closer to that of Portiera than to Hamiltonella in the bacteriocyte. Additionally, Portiera is more ancient than Hamiltonella, so Portiera has a highly reduced genome and has lost its cell wall (36,56,57). The interactions between Vg and Portiera could facilitate the persistence of this symbiont in bacteriocytes. In contrast, Hamiltonella is almost surrounded by Portiera and has an intact cell wall. Hamiltonella may not need the help of Vg for its maintenance in bacteriocytes. Overall, it seems that Hamiltonella uses a different Vg-independent mechanism. Therefore, the Vg symbiosis elucidated in the study is specific only to Portiera. Additionally, there was a higher expression level of Vg in bacteriocytes of female whiteflies than in bacteriocytes of male whiteflies. The effect of Vg on host biology and symbiont abundance in male whiteflies remains unknown. Thus, whether Portiera abundance in the whitefly is governed by a general regulatory mechanism should be tested in males or younger nymphs of whiteflies in future studies.
In this study, we refer to the relative symbiont abundance or density between various treatments. Both the aphid and Buchnera experience polyploidy, which dramatically changes throughout insect development for both players (58,59). Whitefly bacteriocytes and associated symbionts may also show polyploidy as in the aphid-Buchnera system. What have actually been measured here are genome copies of the host and symbiont.

Role of Vitellogenin in Whitefly Symbiosis mBio
Perhaps it would be more conservative to call this symbiont genome copies relative to the insect genome copies. Some studies have shown that Vg delivery for oocyte maturation in the host is coupled with vertical transmission of the symbiont (24,52). We demonstrated that the whitefly and symbiont impact the levels and localization of Vg in bacteriocytes and ovarioles. When the expression of Vg in bacteriocytes and ovarioles was repressed, there was insufficient Vg protein binding to symbiont so that Portiera could be degraded by the immune response. Therefore, silencing Vg reduced the abundance of Portiera in the bacteriocytes and ovarioles of female adult whiteflies. Portiera and Hamiltonella are transmitted into ovarioles via bacteriocyte transfer in whiteflies (21,36). The development of both ovarioles and bacteriocytes impacts the vertical transmission of Portiera in whiteflies. Repressing Vg decreased the number of whitefly ovarioles and eggs, thus inhibiting the transovarial transmission of symbionts. Overall, Vg influences both the abundance and transmission of Portiera in female adult whiteflies. Vg, which is indispensable for oogenesis, seems to gain a novel function involved in symbiont maintenance during long-term evolution of whitefly symbiosis. This study of whitefly symbiosis indicates that Vg is a key player in insect-symbiont interactions and coevolution. It will be interesting to study how Vg impacts the persistence of various insect-microbe symbioses. This information may facilitate our understanding of how host molecules and proteins regulate microbial dynamics.

MATERIALS AND METHODS
Insect rearing and plants. The whitefly B. tabaci MEAM1 colony (mtCO1; GenBank accession no. GQ332577) was maintained on cotton plants (Gossypium hirsutum cv. Shiyuan 321) as described previously (13,34,35). Cotton plants were cultivated to the 6-to 7-true-leaf stage for use in experiments.
Symbiont elimination by antibiotic treatment. To eliminate Portiera, hundreds of adult B. tabaci whiteflies (F0, 0 to 7 days after emergence) were released into each feeding chamber and fed on 25% sucrose solution (wt/vol) supplemented with 30 mg/mL of the antibiotic rifampin (BBI Life Sciences, Shanghai, China) for 2 days as described previously (13). Control whiteflies fed on a sucrose solution not supplemented with antibiotics. Following the antibiotic treatment, B. tabaci whiteflies were transferred to cotton plants. After symbiont quantification by qPCR, the Portiera-cured F1 B. tabaci (2PBt) obtained by antibiotic treatment and control Portierainfected F1 B. tabaci (1PBt) were identified.
qPCR and quantitative reverse transcription-PCR (qRT-PCR). Total DNA was extracted from the whole body, bacteriocytes, and ovaries of female adult whiteflies at day 3 after the whiteflies at day 1 after emergence were microinjected with dsVg in accordance with the Nonidet-P40-based protocol previously described (21). Symbionts were quantified by qPCR using the CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) with 2Â SYBR green master mix (Bimake, Houston, TX, USA). Portiera and Hamiltonella were quantified using symbiont-specific 16S rRNA primers, with the B. tabaci b-actin gene as the internal standard for normalization. All primers used are listed in Table S1 in the supplemental material. Two technical replicates were performed for each of 10 biological replicates for the whitefly whole body and for each of five biological replicates for whitefly bacteriocytes and ovaries after dsVg treatment.
Total RNA was extracted as previously described (34). To compare the expression levels of Vg and VgR in the whole body or bacteriocytes between adult females and adult males and between the whole body and bacteriocytes of adult females or adult males, RNA was extracted from the whole body of 10 female and 10 male adult whiteflies at day 1 after emergence for each of three biological replicates and from bacteriocytes of 50 female and 50 males adult whiteflies at day 1 after emergence for each of three biological replicates. To detect the effect of microinjection with pyriproxyfen or dsJHAMT on the expression of Vg in whiteflies, RNA was extracted from the whole body of 10 female adult whiteflies for each of three biological replicates at days 1 and 2 after the whiteflies at three to four days after emergence were microinjected with pyriproxyfen and from 10 female adult whiteflies for each of three biological replicates at days 5 and 6 after the whiteflies at day 1 after emergence were microinjected with dsJHAMT. To examine the effect of microinjection with dsVg on the expression of Vg in whiteflies, RNA was extracted from the whole body of 10 female adult whiteflies for each of three biological replicates at days 1, 3, and 5 after the whiteflies at day 1 after emergence were microinjected with dsVg. cDNAs were synthesized from the total RNA using an All-in-One cDNA synthesis supermix kit (Bimake, Houston, TX, USA) as described previously (34). The qRT-PCRs were performed using the CFX96 realtime PCR detection system (Bio-Rad, Hercules, CA, USA) with 2Â SYBR green master mix (Bimake, Houston, TX, USA). Relative expression was calculated with the b-actin gene for transcript normalization. All primers used in this study are listed in Table S1. The amplification efficiency of the primers newly designed in this study is high, which is shown in Table S1. Relative symbiont density and relative gene expression were calculated using the 2 2DCT method (60).
FISH. To investigate the localization of Portiera in the bacteriocytes of female adult whiteflies, FISH was conducted in accordance with a previously described protocol (13,34,35).
Immunofluorescence microscopy. Bacteriocytes and/or ovarioles were dissected from 30 female adult whiteflies at day 1 after emergence, female adult whiteflies at various days after dsRNA injection, and Portiera-infected and Portiera-cured adult female whiteflies as well as rapamycin-treated adult female whiteflies for each biological replicate. The samples were fixed, permeabilized, and incubated with Alexa Fluor 488-labeled anti-Vg (42), by following a previously described protocol (13,34). Three biological replicates were conducted. Images were collected and analyzed using an FV3000 confocal microscope (Olympus, Tokyo, Japan). The acquisition parameters were kept constant within the experiment to allow comparison between resulting signal intensities for control/treated whitefly samples.
Immunoelectron microscopy. Bacteriocytes were dissected from adult females (0 to 7 days after emergence), fixed with 3% (vol/vol) paraformaldehyde and 0.2% (vol/vol) glutaraldehyde in 0.2 M phosphate buffer saline overnight at 4°C, dehydrated by a graded ethanol series (30%, 50%, 70%, 90%, and 100%), and embedded in LR Gold Resin (London Resin Company) by referring to a previously described method (36,50,52). Sections were cut at 80 to 100 nm with a Leica EM UC6 ultramicrotome and then blocked for 30 min in blocking buffer (goat serum, 1:100). The blocked sections were incubated at room temperature with the antibodies of anti-Vg mouse serum (1:10) for 2 h and then 10-nm gold-conjugated goat-anti-mouse IgG (1:200; IBM) for 2 h with a wash in distilled water after each antibody incubation. The sections were stained in 2% (wt/vol) neutral uranyl acetate (in distilled water) for 10 min and in lead citrate for 10 to 15 min. The sections were treated without primary antibody and without gold-conjugated goat anti-mouse IgG as the controls. Four biological replicates were conducted. The sections were viewed with a transmission electron microscope (H-7650 Hitachi) at 80-kV accelerating voltage.
Effects of pyriproxyfen injection or silencing of JHAMT on Vg expression and Vg localization. To study the effect of the JH pathway on Vg expression and Vg localization in whiteflies, approximately 800 female adult whiteflies at day 1 after emergence were injected with 1.5 mg/mL dsJHAMT and incubated on cotton leaf disks by using a previously described method (13,45,47,57,61). Control whiteflies were injected with 1.5 mg/mL dsGFP. Whiteflies were collected at 5 and 6 days after dsRNA injection. The survival rates of injected whiteflies were 60% for dsGFP and 40% for dsJHAMT at day 5 after injection. RNA was extracted from the whole body of 10 female adult whiteflies for each of three biological replicates. The expression of JHAMT at days 5 and 6 after dsJHAMT injection and the expression of Vg at day 5 after dsJHAMT injection were examined using qRT-PCR. To detect whether silencing whitefly JHAMT affects Vg localization in ovarioles and bacteriocytes, whiteflies were collected at day 5 after injection. For each biological replicate, ovarioles and bacteriocytes were dissected from 30 female adult whiteflies, fixed, permeabilized, and incubated with antibodies against Vg for ovarioles and bacteriocytes. The samples were incubated with no antibodies against Vg as the negative control. Three biological replicates were conducted. Images were analyzed using an FV3000 confocal microscope (Olympus, Japan). The fluorescence intensity of Vg was analyzed by Image J software. In each of three biological replicates, four ovarioles and three bacteriocytes of dsGFP-injected whiteflies and dsJHAMT-injected whiteflies were used for fluorescence intensity analysis. To further test the effect of JH on Vg expression, approximately 300 female adult wildtype whiteflies at 3 to 4 days after emergence were injected with 0.5 mg/mL pyriproxyfen (JH analog) dissolved in distilled water by using an Eppendorf microinjection system (Hamburg, Germany). Distilled water-injected whiteflies were used as the control. Expression of Vg in female adult whiteflies at days 1 and 2 after the whiteflies were microinjected with pyriproxyfen was examined using qRT-PCR with three biological replicates.
Effects of Portiera elimination by antibiotic treatment on ovariole number and Vg localization in whiteflies. Portiera was eliminated by antibiotic treatment as described above. Following the antibiotic treatment, B. tabaci whiteflies were transferred to cotton plants. F1 female adults were collected. The DNA was extracted from 12 female B. tabaci adults (at 3 to 7 days after eclosion) and used for symbiont quantification by qPCR. To test whether Portiera elimination affects the ovariole number, the number of ovarioles was scored in 10 individuals dissected in PBS at pH 7.4 for 1PBt and -PBt female adult whiteflies (within 4 days after eclosion). To test whether Portiera elimination affects Vg localization in ovarioles and bacteriocytes, ovarioles and bacteriocytes of female B. tabaci adults (at 3 to 7 days after eclosion) were dissected, fixed, permeabilized, and incubated with antibodies against Vg as described above. Three biological replicates were conducted. Images were collected and analyzed on an FV3000 confocal microscope (Olympus, Japan). The fluorescence intensity of Vg was analyzed by Image J software. In each of three biological replicates, three ovarioles and bacteriocytes of 1PBt and -PBt whiteflies were used for fluorescence intensity analysis.
Effects of silencing Vg on Vg localization, symbiont localization and abundance, and whitefly reproduction. To investigate whether silencing Vg influences Vg localization, symbiont localization and abundance, and the numbers of ovarioles and eggs, approximately 2,000 female adult whiteflies infected with Portiera at day 1 after emergence were injected with 1.0 mg/mL dsVg in injection buffer by using an Eppendorf microinjection system (Hamburg, Germany) and incubated on cotton leaf disks as described above. Control whiteflies were injected with 1.0 mg/mL dsGFP. Whiteflies were collected at 3 days after dsRNA injection. The survival rates of injected whiteflies were 75% for dsGFP and 40% for dsVg at day 3 after injection. RNA was extracted from eight female adult whiteflies for each of three biological replicates to examine the expression of Vg 3 days after dsRNA injection. To examine whether silencing whitefly Vg affects Vg localization in ovarioles and bacteriocytes and Portiera localization in bacteriocytes, whiteflies were collected at days 1, 3, and 5 after injection. Whitefly ovarioles and bacteriocytes were dissected, fixed, permeabilized, and incubated with antibodies against Vg for ovarioles as well as antibodies against Vg and a fluorescent probe for Portiera in bacteriocytes as described above. To examine whether silencing whitefly Vg affects Hamiltonella localization in bacteriocytes, whiteflies were collected at day 3 after injection. Whitefly bacteriocytes were dissected, fixed, permeabilized, and hybridized with the fluorescent probe for Portiera and Hamiltonella in bacteriocytes. Three biological replicates were conducted.

Role of Vitellogenin in Whitefly Symbiosis mBio
Images were analyzed using a FV3000 confocal microscope (Olympus, Japan). The fluorescence intensity of Vg and Portiera was analyzed by Image J software. In each of three biological replicates, three ovarioles and bacteriocytes of dsGFP-injected whiteflies and dsVg-injected whiteflies were used for fluorescence intensity analysis of Vg and three bacteriocytes of dsGFP-injected whiteflies and dsVg-injected whiteflies were used for fluorescence intensity analysis of Portiera. To test whether silencing whitefly Vg influences the abundance of symbionts Portiera and Hamiltonella, DNA was extracted from the whole body of individual female adult whiteflies for each of 10 biological replicates and from bacteriocytes and ovaries of eight female adult whiteflies for each of five biological replicates at day 3 after the whiteflies were microinjected with dsVg. Then, qPCR was performed as described above. In parallel, ovarioles were dissected in PBS at pH 7.4, and the number of ovarioles was scored in 10 individuals for dsVg-injected and dsGFP-injected female adult whiteflies at day 3 postinjection. To determine if silencing Vg influences whitefly fecundity, individual Vg-injected and dsGFP-injected whiteflies were transferred onto cotton leaf disks and kept on 1.5% agar plates at 26 6 2°C, with a 14-h-light:10-h-dark photoperiod and 60% to 80% relative humidity (RH). Egg numbers were recorded for the surviving whiteflies. Nineteen biological replicates of individuals were conducted at day 3 postinjection. Western blot analysis. Whiteflies at day 1 after emergence were injected with 1.0 mg/mL dsVg and 1.0 mg/mL dsGFP in injection buffer as described above. In total, 50 dsGFP-injected female adult whiteflies and 50 dsVg-injected female adult whiteflies 3 days after dsRNA microinjection were collected. Proteins were extracted, separated by 12% SDS-PAGE, and then blotted by following standard procedures using previously described protocols (13,47). Primary antibodies specific for the following proteins were used for Western blotting: Vg (42) and b-actin. The secondary antibody used was goat anti-mouse antibody for Vg (Beyotime Biotechnology, Shanghai, China). Three biological replicates were conducted. The signal was detected using a ChemiDoc XRS1 (Bio-Rad, Hercules, CA, USA). The densitometry for protein levels was analyzed using Image J software.
IC-PCR assay. The interaction between Vg and Portiera was examined using an IC-PCR assay by previously described protocols (29). PCR tubes were coated with 25 mL antibody against Vg (1:1,000 diluted in coating buffer), for 1.5 h at 37°C, and then washed five times for 5 min each time with 50 mL washing buffer. Homogenates of bacteriocytes and heads that were collected from 8 to 10 whiteflies in 5 mL PBS were incubated for 18 h at 4°C in the coated PCR tubes. The tubes were washed five times, 5 min each time, with 25 mL washing buffer and dried. PCR amplification of Portiera bound to the Vg protein, which was itself bound to the antibody-coated tubes, was performed with Portiera-specific 16S rRNA gene fragment primers (Table S1). The control no-antibody-coated tubes were incubated with a homogenate of the bacteriocytes and heads of whiteflies, and antibody-coated tubes incubated with a homogenate of whitefly heads not containing bacteriocytes served as controls.
Statistical analyses. For the mRNA expression level, fluorescence intensity, protein level, and symbiont abundance, as well as the numbers of Vg gold dots, ovarioles, and eggs, statistical differences were evaluated using one-way analysis of variance (ANOVA) at a significance threshold of 0.05. All of the data analyses were conducted using STATISTICA v6.1 software (StatSoft, Inc., Tulsa, OK, USA).
Ethical approval and consent to participate. The insects used in this study are agricultural pests. No ethics approval is needed.
Data availability. All data generated or analyzed during this study are included in the article and the supplemental material.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.