The Impact of Host Diet on Titer in

Summary

Many invertebrate organisms carry bacterial endosymbionts within their cells. In many cases, this ensures host access to resources provided by the endosymbionts, and reciprocally, a rich source of host-supplied nutrients supports bacterial growth and reproduction. However if bacterial reproduction is uncontrolled, an over-abundance of bacteria will ultimately destroy the host cell. Here we explore the factors that regulate endosymbiont abundance in host cells. We focused on Wolbachia endosymbionts that are carried naturally in the germ cells of fruit flies. Specifically, we determined whether dietary nutrients affect the amount of Wolbachia bacteria carried by female flies. We found that yeast-enriched diets strongly depleted Wolbachia in fly ovarian cells. By contrast, sucrose-enriched diets doubled the amount of Wolbachia in ovarian cells. In addition, we found that this response to diet is mediated through highly conserved TORC1 and insulin signaling pathways in the fly. Recent studies have revealed that host diet dramatically influences the types and abundance of gut microbes. Our study informs how host diet affects endosymbiotic bacteria housed within specific types of host cells.

Introduction

Microbial endosymbionts have a profound impact on host metabolism and there are numerous examples in which microbes provide essential nutrients to the host [1–14]. In contrast, considerably less is known regarding how host metabolism and nutrition affect resident endosymbionts. To date, there is evidence that restricting the supply of host carbon, nitrogen and phosphorous significantly limits the number of Chlorella endosymbionts of green hydra and dinoflagellate endosymbionts of cnidarians [1]. Researchers have also observed that exposure to high levels of exogenous thiamine monophosphate suppresses the titer of Sodalis and Wigglesworthia endosymbionts in tsetse flies [15,16]. In this largely unexplored area, many outstanding questions remain: What are the host and endosymbiont metabolic and signaling pathways involved in nutrient sensing? To what extent do endosymbionts exhibit tissue-specific responses to nutrient availability? How are the rates of endosymbiont replication and cell death influenced by host metabolism and nutrients?

The symbiosis between Wolbachia and Drosophila is an excellent system to experimentally address these issues. Wolbachia are obligate intracellular endosymbionts carried by an estimated 40% of all insect species, including the established model organism Drosophila melanogaster [17–20]. Though Wolbachia endosymbionts are naturally carried within germline cells of both male and female insects, Wolbachia are ultimately removed from sperm prior to completion of spermatogenesis [17,18,21–25]. Thus, Wolbachia rely upon transmission through the maternal germline for their success. In addition to its functional importance in Wolbachia transmission, the well-characterized molecular and cell biology of Drosophila oogenesis has provided considerable contextual information and experimental tools that can be applied to studies of Wolbachia-host interactions [18,26–30].

The primary developmental units of the ovary that carry Wolbachia are referred to as egg chambers [27,28]. In each egg chamber, an outer layer of somatic follicle cells encapsulates an interconnected cyst of germline cells, comprised of 15 nurse cells and an oocyte. Wolbachia are initially loaded into these developing cysts during the first mitotic division from a Wolbachia-infected germline stem cell [18,31]. This germline Wolbachia population is amplified over time by binary fission and likely to some extent by exogenously invading Wolbachia [31–36]. Wolbachia persist in the germline throughout oogenesis, and a subset of the bacteria concentrate at the oocyte posterior pole during mid- to late oogenesis [31,37,38]. This ensures incorporation of Wolbachia into germline progenitor cells that form at the embryonic posterior pole, perpetuating the maternal germline transmission cycle [39]. Thus, maintenance of a sufficient Wolbachia titer in germline cells is important for success of the germline-based transmission strategy.

Here we examined how host diet affects Wolbachia titer in Drosophila melanogaster. The data demonstrate that yeast-enriched diets suppress Wolbachia titer and lead to altered nucleoid morphology during oogenesis. Genetic and chemical disruptions indicate that the somatic insulin and TORC1 pathways (Fig. 1) are required for yeast-based suppression of oocyte Wolbachia titer. The data also indicate that sucrose-enriched diets increased oocyte Wolbachia titer, with little impact on nucleoid morphology. Evidence indicates that yeast-enriched diets substantially increase somatic Wolbachia titers, though this was not the case in the central nervous system (CNS). These studies demonstrate that Wolbachia, and likely other bacterial endosymbionts, exhibit distinct, tissue-specific responses to host nutrients that involve conserved signaling and metabolic pathways.

Results

Exposing Drosophila to a yeast-enriched diet suppresses germline Wolbachia titer

Nutrient availability strongly affects the life cycle of cultured bacteria, raising questions about how host nutrient conditions affect intracellular Wolbachia bacteria. As D. melanogaster in nature preferentially consume yeast [40–45], we tested the effect of dietary yeast on Wolbachia titer in vivo. Female flies were aged first for two days on standard food, then fed yeast paste for 3 days, and examined for Wolbachia titer in oogenesis. Ovarian tissues were stained with propidium iodide to label Wolbachia DNA, and the Wolbachia nucleoids imaged in oocytes of stage 10 egg chambers by confocal microscopy [38]. This analysis demonstrated that yeast paste-fed oocytes carried far less Wolbachia than control oocytes (Fig. 2A-B) (S1 Table). Wolbachia were further quantified within single oocyte focal planes to determine relative titer for each condition [32]. This revealed that Wolbachia titer in yeast paste-fed oocytes was at 27% of the control level. Oocytes treated with standard fly food exhibited an average of 229 +/- 21.1 Wolbachia puncta (n = 30), as compared to yeast paste-fed oocytes that carried 62.6 +/- 4.33 Wolbachia (n = 29) (p < 0.001) (Fig. 2C). This indicates that host exposure to yeast paste significantly reduces Wolbachia titer in oogenesis.

One possibility is that yeast paste diets reduce oocyte titer because other critical nutrients provided by standard fly food are unavailable. To address this issue, 2-day old Drosophila were fed with either standard food diluted 1/3 with water, thereafter referred to as “control food”, or fed with standard food diluted 1/3 with yeast paste, thereafter referred to as “yeast-enriched food” (S1 Table). After 3 days of exposure to these conditions, titer was assessed in oogenesis. The yeast-enriched condition exhibited 55% of the control titer level, with controls displaying 124 +/- 10.8 Wolbachia (n = 58), compared to yeast-enriched oocytes carrying 68.7 +/- 5.12 Wolbachia (n = 35) (p = 0.001) (Fig. 2D). To further assess whether this is due to differences in food hydration between control and yeast-enriched conditions, we also exposed flies to a 1/3 dilution of corn syrup into standard fly food (S1 Table). Although corn syrup-enriched food is less hydrated than control food, it resulted in similar oocyte titer measurements as the control, with an average of 128 +/- 12.9 Wolbachia visible per oocyte (n = 31) (Fig. 2D). These data together suggest that yeast-induced titer reduction is not due to depletion of specific nutrients or water available in standard food. Rather, the data indicate that dietary yeast is responsible for reducing Wolbachia titer carried by oocyte cells.

To determine whether dietary yeast can induce a similar oocyte titer response in wild insects as seen in laboratory fly stocks, Drosophila melanogaster and Drosophila simulans were collected from nature. These flies were exposed to yeast-enriched food and assessed for Wolbachia titer in oogenesis. We found that oocyte Wolbachia titer in the yeast-enriched condition was at 47% of the control level, with an average of 94.8 +/- 21.8 Wolbachia detected in control oocytes (n = 12), versus 44.6 +/- 6.52 Wolbachia detected in the yeast-enriched condition (n = 13) (p = 0.029) (S1 Fig). Thus, yeast-enriched diets suppress oocyte Wolbachia titer in wild-caught Drosophila analogous to laboratory D. melanogaster strains.

To further investigate the basis for yeast-associated Wolbachia depletion in oocytes, Wolbachia titer was examined in the germline-derived nurse cells associated with the oocyte. It is currently unclear in Drosophila when or how frequently Wolbachia travel through the ring canals between the nurse cells and oocyte. Thus, it is possible that Wolbachia depletion in oocytes could be due to preferential retention in the nurse cells. To investigate this, we imaged Wolbachia in equivalent focal planes of nurse cells and oocytes within single egg chambers and analyzed their Wolbachia titer [32]. Overlaid images showing a planar reconstruction of egg chambers indicated fewer Wolbachia throughout the germline cells of yeast-exposed organisms (Fig. 3A-B). Quantitation of the yeast-enriched condition indicated that nurse cells carried 27% of the control titer level (Fig. 3C). Specifically, 52.6 +/- 4.93 Wolbachia per nurse cell were detected in the control (n = 20), in contrast to 14.4 +/- 1.65 Wolbachia per nurse cell in the yeast-enriched condition (n = 20) (p < 0.001) (Fig. 3C). Furthermore, oocyte titer in the yeast-enriched condition was 14% of the control level, with 420 +/- 44.6 Wolbachia detected in control oocytes (n = 17), versus 59.0 +/- 11.1 Wolbachia in oocytes from the yeast-enriched condition (n = 20) (p < 0.001) (Fig. 3D). These data indicate that Wolbachia redistribution between germline cells is not responsible for the low oocyte titer observed in yeast-exposed organisms. Rather, yeast-enriched food induces similar Wolbachia depletion in nurse cells and oocytes.

The nutrient-responsive kinase complex, TORC1, affects oocyte Wolbachia titer

Cells coordinate intracellular events in response to exogenous nutrients using multiple signaling pathways that converge upon the Target of Rapamycin kinase complex 1 (TORC1) (Fig. 1) [46]. TORC1 can be activated by an amino-acid dependent signaling mechanism, or by insulin signaling (Fig. 1) [46–48]. To test whether TORC1 activity affects oocyte Wolbachia titer, flies were exposed to standard food containing the TORC1 inhibitor, rapamycin [49–52]. This experiment indicated that rapamycin treatment drove a 1.7-fold increase in oocyte Wolbachia titer (Fig. 4A). The average titer from control oocytes, exposed to DMSO-containing standard food, was 207 +/- 22.1 Wolbachia (n = 28). By contrast, oocytes exposed to rapamycin-containing standard food had 357 +/- 31 Wolbachia (n = 30) (p < 0.01) (Fig. 4A). Since rapamycin exposure leads to higher oocyte Wolbachia titer, this suggests that a normal consequence of TORC1 activity is suppression of oocyte Wolbachia titer.

If TORC1 function normally leads to decreased oocyte Wolbachia titer, then hyper-activation of TORC1 would be expected to drive a further reduction of oocyte titer. Branched chain amino acids (BCAAs) taken up through the Slimfast transporter can induce up-regulation of TORC1 (Fig. 1) [53–58]. Therefore, we fed flies a slurry of BCAAs diluted 1/3 into standard food (S1 Table), and assessed Wolbachia titer in oogenesis. Wolbachia titer in the BCAA condition was reduced to 77% of the control (Fig. 4B). This was indicated by an average of 137 +/- 9.71 Wolbachia in control oocytes (n = 34) versus 105 +/- 8.48 Wolbachia in oocytes from the BCAA condition (n = 33) (p = 0.015) (Fig. 4B). The data suggest that TORC1 stimulation with BCAAs drives oocyte titer reduction, opposite the effects of the TORC1 inhibitor, Rapamycin.

To further investigate a possible role for TORC1, we genetically manipulated a key regulator of TORC1 activity. Tsc2, known as Gigas in Drosophila, is downstream of the insulin receptor (Fig. 1) [59–64]. If Tsc2 function is suppressed by any means, this allows TORC1 to become active (Fig. 1) [46,64–68]. Therefore, we tested the impact of Tsc2 on oocyte Wolbachia titer by expressing Tsc2 dsRNA under the control of germline- and soma-specific GAL4 drivers [69–72]. This investigation revealed different oocyte Wolbachia titer responses to tissue-specific Tsc2 RNAi knockdowns. Our efforts to manipulate Tsc2 dosage in germline cells had no impact on oocyte titer (Fig. 4C). An average of 182 +/- 13.5 Wolbachia were detected in control oocytes (n = 53), which was not significantly different from the 207 +/- 17.7 Wolbachia detected in response to germline Tsc2 RNAi (n = 56) (Fig. 4C). By contrast, Tsc2 RNAi knockdowns in the somatic cells reduced oocyte Wolbachia titer to approximately 50% of the control level (Fig. 4D). Control oocytes exhibited an average of 402 +/- 43.4 Wolbachia (n = 24). However, oocytes somatic Tsc2 knockdown flies exhibited an average of 181 +/- 19.8 oocyte Wolbachia (n = 21) (p < 0.001) (Fig. 4D). As such, these data implicate somatic Tsc2, and thus somatic TORC1 signaling, in regulation of oocyte Wolbachia titer.

Yeast suppression of oocyte Wolbachia titer is mediated by insulin-TORC1 signaling

A role for somatic TORC1 in regulating oocyte Wolbachia titer raised the question of whether dietary yeast stimulates TORC1. This could occur through either protein- or insulin-based mechanisms (Fig. 1). As yeast is major source of protein for D. melanogaster, perhaps its amino acid content stimulates TORC1 to ultimately suppress oocyte Wolbachia titer. To test this possibility, we exposed flies to food enriched in Bovine Serum Albumin, prepared specifically to match the protein content of yeast-enriched food (S1 Table). Oocyte Wolbachia titer was similar for control and BSA-enriched conditions, however, with the control exhibiting 1260 +/- 102 Wolbachia (n = 26), and the BSA-enriched condition exhibiting 1190 +/- 48.2 Wolbachia (n = 18) (S2 Fig). This suggests that amino acid availability in the host diet has little impact on oocyte Wolbachia titer.

An alternate possibility is that yeast-enriched diets affect oocyte Wolbachia through insulin stimulation of TORC1. It was previously shown that dietary yeast stimulates insulin-producing cells (IPCs) the brain to release the insulin-like-peptides (Dilps) into the hemolymph [73,74]. To test whether yeast acts through somatic Dilp secretion to oocyte Wolbachia titer, we ablated the IPCs in the brain of fully mature Drosophila females. This is achieved using a dilp2: Gene-Switch-GAL4, UAS: Reaper system that specifically kills off the brain IPCs in response to a 2-week mifepristone treatment [74].

We first investigated whether mifepristone on its own modulates the yeast effect in wild-type flies. After completing a two-week exposure to either DMSO or mifepristone, flies were exposed to either control or yeast-enriched food for 3 days, and their oocyte titer levels were assessed. DMSO-treated flies exhibited substantial oocyte titer depletion in response to yeast-enriched food, down to 30% of the titer in the control condition (Fig. 5A). This was indicated by 785 +/- 64.8 Wolbachia per oocyte in the DMSO-control food condition (n = 24), in contrast to 191 +/- 26.9 Wolbachia in the DMSO-yeast-enriched condition (n = 25) (p <. 001) (Fig. 5A). Mifepristone-treated flies showed a similar titer reduction after exposure to yeast, exhibiting 21% of the titer seen in the control food condition (Fig. 5B). This was indicated by 896 +/- 77.2 Wolbachia per oocyte in the mifepristone-control food condition (n = 23), versus 264 +/- 39.5 Wolbachia in the mifepristone-yeast-enriched condition (n = 25) (Fig. 5B) (p <. 001). Therefore, mifepristone alone has no effect on yeast-based suppression of oocyte Wolbachia titer.

Next, the exact same treatment regimens were performed on flies with the dilp2: Gene-Switch-GAL4, UAS: Reaper genotype. In this experiment, DMSO-treated flies, which retained functional IPCs, exhibited a severe oocyte Wolbachia depletion in response to yeast-enriched food, exhibiting only 7% of the oocyte titer seen on DMSO-control food (Fig. 5C). This was indicated by the presence of 999 +/- 116 Wolbachia per oocyte in the DMSO-control food condition (n = 17), versus 66.5 +/- 6.61 Wolbachia in the DMSO-yeast-enriched condition (n = 20) (p < 0.001) (Fig. 5C). In stark contrast, mifepristone-treated flies that had lost their IPCs exhibited no oocyte titer change after exposure to yeast (Fig. 5D). This was indicated by detection of 583 +/- 72.6 Wolbachia per oocyte in the mifepristone-control food condition (n = 20), versus 503 +/- 68.0 Wolbachia in the mifepristone-yeast-enriched condition (n = 20) (Fig. 5D). Since mifepristone in combination with the dilp2: Gene-Switch-GAL4, UAS: Reaper system specifically prevented yeast from affecting oocyte Wolbachia titer, this demonstrates that somatic IPCs mediate Wolbachia titer suppression by dietary yeast.

Dietary sucrose elevates oocyte Wolbachia titer in an insulin-dependent manner

To further investigate the sensitivity of oocyte Wolbachia titer to somatic insulin signaling, we also examined the effect of a sucrose-rich, high sugar diet. High sugar diets have been shown to induce insulin resistance in Drosophila [75,76]. This is may be due in part to increased expression of NLaz [75], which in mammals is known to suppress Akt function within the insulin signaling pathway (Fig. 1) [77–79]. To test the impact of sucrose-enriched diets on oocyte Wolbachia titer, 2-day old D. melanogaster were fed standard food diluted 1/3 with saturated sucrose solution, hereafter referred to as “sucrose-enriched food” (S1 Table). After 3 days of exposure to this diet, Wolbachia titer was assessed in oogenesis. Oocytes from the sucrose-enriched condition exhibited a 2.4-fold increase in Wolbachia (Fig. 6A). Unlike oocytes raised on control food, which exhibited an average of 165 +/- 22.2 Wolbachia (n = 24), D. melanogaster oocytes exposed to sucrose-enriched food exhibited 392 +/- 25.3 Wolbachia (n = 26) (p < 0.001) (Fig. 6A). These data indicate that a high sugar diet significantly elevates oocyte Wolbachia titer, possibly via an insulin-related mechanism.

A sucrose-based impact on oocyte Wolbachia titer is surprising, as corn syrup-enriched food did not induce a similar effect (Fig. 2D). Notably, sucrose is a disaccharide, composed of glucose and fructose, whereas corn syrup consists mainly of glucose. To elucidate the basis for sucrose-induced titer effects in oogenesis, food enriched for glucose and fructose were also tested. However, none of the monosaccharide-enriched conditions significantly affected oocyte Wolbachia titer (Fig. 6B). Control food yielded an average oocyte titer of 478 +/- 27.6 Wolbachia per oocyte (n = 71). Similarly, oocytes in the glucose-enriched condition displayed 520 +/- 31.1 bacteria (n = 33), the fructose-enriched food condition resulted in 478 +/- 33.0 Wolbachia (n = 29), and a mixture of glucose + fructose yielded 499 +/- 28.0 Wolbachia (n = 32). By contrast, oocytes from the sucrose-enriched condition presented 883 +/- 95.4 Wolbachia (n = 22) (p <. 001) (Fig. 6B). This confirms that disaccharide sucrose molecule specifically elicits Wolbachia titer increases in oogenesis.

To further test the possibility that insulin signaling mediates sucrose impact on ovarian Wolbachia titer, we coupled genetic disruptions of the insulin pathway with sucrose-enriched food. Chico is a Drosophila homolog of the Insulin Receptor Substrate that relays signals from the Insulin Receptor to AKT kinase, and thus ultimately TORC1 (Fig. 1) [80,81]. Germline and soma-specific GAL4 drivers were used to drive expression of chico dsRNA [69–72], and oocyte Wolbachia titer was assayed in control and sucrose-enriched conditions. This test did not indicate any effect of germline chico RNAi on sucrose-induced oocyte titer elevation, with sucrose-enriched food corresponding to 2.4-fold higher oocyte titer than the control (Fig. 6C). Germline chico RNAi oocytes exhibited 125 +/- 10.6 Wolbachia when exposed to regular food (n = 26) as compared to 299 +/- 27.2 Wolbachia in response to sucrose-enriched food (n = 19) (p < 0.001) (Fig. 6C). By contrast, somatic chico RNAi eliminated sucrose-induced titer effects in oogenesis (Fig. 6D). Oocytes from somatic chico RNAi flies exhibited 180 +/- 12.9 Wolbachia in the control condition (n = 25), as compared to 169 +/- 12.5 Wolbachia per oocyte in the sucrose-enriched condition (n = 25) (Fig. 6D). Analysis of sibling controls further indicated that the genetic background for the somatic chico RNAi experiment was not responsible for differential oocyte titer responses to sucrose (Fig. 6E). In flies carrying the somatic da-GAL4 driver used for this experiment, the sucrose-enriched condition continued to exhibit 2-fold more Wolbachia than the control food condition. An average of 124 +/- 11.1 Wolbachia were detected in control oocytes (n = 27) as compared to 251 +/- 32.8 Wolbachia detected in oocytes from the sucrose-enriched condition (n = 20) (p <. 001) (Fig. 6E). Though the complete mechanistic implications of somatic chico disruption remain unclear, these data demonstrate that sucrose acts through somatic insulin signaling to elevate oocyte Wolbachia titer.

Oocyte Wolbachia titer responses are independent of ovary productivity

These data raise the fundamental question of why diet-modulated insulin signaling affects Wolbachia titer so strongly in germline cells. One possibility is that these titer responses are an indirect result of nutrient-induced changes in ovary size and productivity [76]. Yeast-rich diets and insulin signaling are known to drive formation of larger, more productive ovaries [60,76,80,82–91], while high-sucrose diets have the opposite effect [76–79]. To test the contribution of ovary size and productivity variables on oocyte Wolbachia titer, we manipulated ovary productivity by controlling female mating. Mating stimulates ovary development, resulting in a moderately sized, productive ovary. By contrast, virgin females exhibit very large ovaries, filled mainly by mature eggs [92–96]. Oocytes from mated versus virgin females revealed similar oocyte Wolbachia titers, however (S3 Fig). The mated condition displayed 449 +/- 27.5 Wolbachia per oocyte (n = 26), while the virgin female condition that carried 470 +/- 40.6 Wolbachia per oocyte (n = 24) (S3 Fig). These data suggest that ovary size and productivity do not serve as the primary determinants of oocyte Wolbachia titer.

Wolbachia nucleoid morphology responds to dietary yeast

To further investigate the effects of host diet on Wolbachia, we examined Wolbachia nucleoid morphology. Other studies indicate that nucleoid morphology can serve as a proxy indicator of replication-associated changes in cell shape, or stress-induced DNA compaction [97–99]. Multiple, zoomed-in images of Wolbachia stained with propidium iodide were projected as a single image, and nucleoid shape was measured. The images indicated that Wolbachia nucleoid shape differs between nutrient conditions (S4 Fig). To specifically analyze changes in nucleoid length, 120 nucleoids were selected at random from each treatment condition and their lengths were compared. This analysis indicated that 50% of nucleoids in the control condition exceeded 2 μm in length (S4 Fig). The sucrose-enriched condition was similar, with 53% of nucleoids exceeding 2 μm. In the yeast-enriched condition, however, only 37% of nucleoids exceeded this measure (p <. 05). Thus, yeast-enriched food significantly shortened Wolbachia nucleoids. We further determined an elongation index (EI), representing bacterial length divided by width, for the same 120 nucleoids per treatment condition as above. This analysis indicated that 50% of nucleoids measured in the control condition had an EI greater than 2. In the sucrose-enriched condition, only 33% of nucleoids showed an EI greater than 2 (p <. 05). In the yeast-enriched condition, even fewer nucleoids showed this degree of elongation, with only 22% of nucleoids exceeding this EI (p <. 001) (S4 Fig). These data indicate that dietary conditions, and especially exposure to yeast-enriched food, alter Wolbachia nucleoid morphology in oogenesis. This is consistent with a bacterial physiological response to host diet.

Wolbachia titers are regulated in a tissue-specific manner

The striking impact of dietary nutrients on oocyte Wolbachia titer raises the question of whether Wolbachia titer in other tissues is responsive to nutrient conditions. Wolbachia are present in insect somatic cells, and the Drosophila brain is particularly amenable to assessment of somatic Wolbachia titer [100,101]. To take advantage of this, we imaged Wolbachia in the central brain of D. melanogaster exposed to different nutrient conditions. This analysis revealed that D. melanogaster on control food already carry very low Wolbachia titer in the central brain (Fig. 7A, A’, n = 3), and flies fed with either yeast-enriched or sucrose-enriched food were indistinguishable in appearance from the control (Fig. 7B, B’, n = 3) (Fig. 7C, C’, n = 3). Thus, Wolbachia titer in D. melanogaster brain does not appear to be affected by the dietary conditions used in this study. An alternative possibility, however, is that the overall low Wolbachia titer detected under these conditions hampered our ability to assay nutrient-induced changes in titer.

To pursue this further, the impact of nutrient-altered food was tested in the closely related D. simulans species, known for carrying high Wolbachia titer in its brain cells [101]. Flies exposed to control food exhibited a high titer of Wolbachia in the central brain overall (Fig. 7D, D’, n = 7). Similarly high Wolbachia titer was detected in the brain after exposure to yeast-and sucrose-enriched food (Fig. 7E, E’, n = 5) (Fig. 7F, F’, n = 4). Further quantification of Wolbachia infection frequency did not reveal any differences between nutrient conditions (Fig. 7G). In control food, yeast-enriched, and sucrose-enriched conditions, 55–56% of brain cells exhibited Wolbachia infection (n = 1171, 767, and 665 cells, respectively). No differences were seen in formation of large Wolbachia aggregates either (Fig. 7H). Brain samples reared on control food, yeast-enriched, and sucrose-enriched conditions all exhibited between 16–19 large bacterial clusters per hundred cells. This indicates that Wolbachia titer in the D. simulans brain is unresponsive to the nutrient-altered conditions used in this study.

To address the possibility that D. simulans tissues are generally unresponsive to nutrients, we also assessed D. simulans oocyte titer in response to nutrient-altered food. In contrast to the brain, D. simulans oocytes exhibited a clear nutrient-dependent Wolbachia titer response (S5 Fig). Control oocyte images carried 293 +/- 49.9 Wolbachia (n = 10). By contrast, oocyte titer from the yeast-enriched condition was at 40% of the control level, with an average of 116 +/- 20.1 bacteria detected per oocyte (n = 10) (p = 0.004). Furthermore, the sucrose-enriched condition exhibited 2.3-fold higher titer than the control, with 662 +/- 73.6 Wolbachia detected per oocyte (n = 10) (p = 0.001) (S5 Fig). Thus, D. simulans Wolbachia titers are capable of responding similarly to nutrient conditions as D. melanogaster.

To further probe the impact of host diet on somatic Wolbachia titer, we analyzed relative amounts of Wolbachia versus host DNA in ovarectomized female flies. In this analysis, females were exposed to nutrient-altered diets, dissected to remove ovarian tissues, and analyzed by qPCR. The results indicate the relative level of Wolbachia per host genome copy number. This analysis indicated that yeast-enriched dietary conditions led to higher levels of Wolbachia than the control food condition (Fig. 7I). Control samples exhibited a mean relative level of Wolbachia of 0.989 (n = 37), whereas the yeast-enriched condition displayed a mean relative level of Wolbachia of 1.28 (n = 35) (p < 0.05). Females exposed to sucrose-enriched diets were not significantly different from the control, however, exhibiting a mean Wolbachia relative level of 0.792 (n = 36) (Fig. 7I). This titer response profile differs from analyses of Wolbachia titer in the ovary as well as the brain. This suggests that host diet affects Wolbachia titers in a tissue-specific manner.

As host nutrition has a different impact on ovarian versus somatic Wolbachia titers, this raises the question of what would happen in organism lacking ovarian tissue altogether. To address this issue, qPCR analysis was performed on intact male flies. This indicated that bodywide Wolbachia titer also increases in response to yeast-enriched food, although not sucrose-enriched food (Fig. 7J). The control food condition carried a mean Wolbachia relative level of 1 (n = 16), in contrast to the yeast-enriched condition, which displayed a mean Wolbachia relative level of 1.545 (n = 15) (p < 0.05). Sucrose-enriched diets corresponded to a mean Wolbachia relative level of 1.027 (n = 16). This analysis confirms that the profile of bodywide titer responses in males is equivalent to ovarectomized females. This suggests that somatic Wolbachia titers overall respond to host dietary conditions in a consistent manner.

Discussion

The major finding of this study is that dietary intake by Drosophila strongly influences Wolbachia titer in the host female germline: a high yeast diet decreases Wolbachia oocyte titer and a high sucrose diet increases Wolbachia oocyte titer. This finding adds to a small but growing literature on the impact of host diet on endosymbionts [1,15,16]. Prior studies of Wolbachia suggest that this endosymbiont relies heavily upon host provisioning of amino acids and carbohydrates [102–104]. A very recent study analyzing the Drosophila midgut and ovary surprisingly indicated that neither dietary yeast nor sucrose had any affect on the Wolbachia:host genomic ratio in those tissues [105]. The image-based analyses of this study demonstrate that yeast and sucrose affect germline Wolbachia titer at the cellular level, however. It is unclear why Wolbachia titer in the oogenesis should be particularly sensitive to diet and whether this is an adaptive response to changes in the host metabolic environment. The evolutionary success of Wolbachia depends on its ability to localize at the posterior pole of the oocyte, the site of germline formation. Significantly, we find that Wolbachia localize to the posterior pole regardless of whether the host is exposed to the low titer, yeast-enriched diet, or the high titer, sucrose-enriched diet. This suggests the previously described microtubule and motor protein based mechanisms driving posterior localization of Wolbachia [38] are robust, even in the face of dramatic titer changes caused by nutrient-altered diets.

Insight into the mechanism of yeast-induced titer suppression comes from our functional studies demonstrating that this response is mediated through TORC1. Genetic up-regulation of TORC1 suppresses oocyte Wolbachia titer, whereas drug-based inhibition of TORC1 increases titer. This finding creates the basis for a sensible functional connection between intracellular Wolbachia and host diet, as both amino acids and insulin signaling are known to drive TORC1 activity [46]. Our finding that BSA-enriched food had no effect on oocyte Wolbachia titer argues that yeast protein content is not the major determinant of germline titer suppression, and alternatively suggests a role for insulin signaling. Prior work has shown that yeast-rich diets trigger insulin signaling in Drosophila, and that Wolbachia interact with host insulin signaling processes [89,106]. Our finding, that loss of somatic IPCs eliminates yeast impact on oocyte Wolbachia titer, confirms that insulin signaling facilitates the titer-suppressing effects of yeast. Furthermore, disrupting the somatic insulin receptor substrate, Chico, suppressed the impact of dietary sucrose on oocyte Wolbachia titer. This suggests that both dietary yeast and sucrose affect germline Wolbachia titer via antagonistic impacts on somatic insulin signaling (Fig. 8).

In considering the mechanism of insulin-based impact on germline Wolbachia titer, one possibility is that changes in ovary productivity are responsible. Diet-modulated insulin signaling affects the relative rates of germline stem cell division, germline cell survival and egg chamber development [60,76,80,82–91]. If Wolbachia are unresponsive to nutrient-induced adjustments in germline cell growth and development, significant titer changes in oogenesis would be expected. However, oocyte Wolbachia titers were very similar in mated and virgin females, despite the different rates of germline stem division expected for each type of flies [76,83,86,88,90–96]. Another possibility is that yeast-induced insulin signaling affects Wolbachia physiology in oogenesis. The “rounded” Wolbachia nucleoids visible in the yeast-enriched condition could indicate substantially slowed bacterial growth or a bacterial stress response, for example [97–99]. Insulin signaling has been shown to induce changes in cytoskeleton organization, proteasome activity and chaperonin activity [107–111], any of which could affect Wolbachia physiology. It is also possible that dietary yeast in particular carries one or more bioreactive agents that are toxic to germline Wolbachia (Fig. 8).

The impact of somatic insulin signaling on germline Wolbachia titer also raises the question of whether somatic Wolbachia titers are similarly affected by host nutrient conditions. Our initial findings that Wolbachia titers in the Drosophila brain are non-responsive to host diet suggested that nutrient-associated titer changes are restricted to the ovary. Analysis of sucrose-fed, ovarectomized females is further consistent with that interpretation. However, analysis of ovarectomized females also indicated that dietary yeast triggers somatic titer changes opposite of oogenesis. It is possible that this occurs by physical relocation of Wolbachia within the body, with dietary yeast driving Wolbachia egress from ovarian cells, followed by invasion of somatic target tissues. Alternatively, host dietary conditions may drive tissue-specific differences in the Wolbachia life cycle. Perhaps yeast-enriched diets favor Wolbachia replication and survival in specific somatic tissues while disfavoring the same in oogenesis. Support for this hypothesis comes from our finding that yeast-enriched food induces the same bodywide titer changes in male flies as seen in ovarectomized females. This demonstrates that ovarian Wolbachia titer responses are distinct from that of other tissues.

The pathways downstream and upstream of TORC1 that mediate yeast-based suppression of Wolbachia germline titer are yet to be determined. An obvious possibility is the role of TORC1 in suppressing autophagy (Fig. 8). There are numerous examples in which autophagy either enhances or suppresses intracellular bacteria titer [112]. Since TORC1 disruptions increase Wolbachia titer in oogenesis, it is possible that Wolbachia interact positively with autophagy, consistent with other endosymbionts [113] [114]. As insulin signaling is expected to down-regulate autophagy (Fig. 1), the low Wolbachia titers seen in yeast-fed oocytes are further consistent with this possibility. However, the finding that dietary yeast also increases somatic Wolbachia titers implies that somatic autophagy is normally bactericidal in that context, consistent with another recent report [115]. These conflicting results may indicate that tissue-specific differences in autophagy regulation contribute to Wolbachia titer control, or that other mechanisms downstream or independent from autophagy are responsible (Fig. 8). Perhaps responses from one or more other TORC1 effectors further contribute to Wolbachia titer regulation (Fig. 1).

Wolbachia have been shown to suppress replication of RNA viruses in insects, including the human pathogens, Dengue Fever Virus and Chikungunya Virus [116–118]. This finding, together with the fact that Wolbachia-induced Cytoplasmic Incompatibility rapid spreads Wolbachia through insect populations [25,119], has led to a novel strategy of combating these diseases by releasing Wolbachia-infected insect carriers of these viruses into afflicted regions [120,121]. Although the mechanism of Wolbachia-induced viral suppression is unknown, several studies demonstrate that the higher the Wolbachia titer, the greater the viral suppression [122–126]. Our finding that host diet dramatically affects tissue-specific Wolbachia titers suggests that the natural diets of the released insects should be taken into account when evaluating the potential effectiveness of a Wolbachia-based viral suppression field study. Finally it will be of interest to determine whether diet has a similar effect on Wolbachia titer in disease-associated filiarial nematodes.

Materials and Methods

Fly strains

Natural D. melanogaster and D. simulans flies were harvested daily from collection buckets distributed in the Santa Cruz, CA area. As the female flies of these species are morphologically indistinguishable, but both species were well-represented in the area, this wild-caught population was presumed to represent both species. The laboratory strain of D. simulans used was a w- stock that carried the endogenous wRi Wolbachia strain. The D. melanogaster strain used for the initial nutrient feeds and for crossing wMel Wolbachia into the other fly strains was w; Sp/Cyo; Sb/TM6B. Other D. melanogaster fly strains used were the gigas VALIUM20 TRiP line: y, sc, v; P{TRiP.HMS01217}attP2/TM3, Sb; the chico VALIUM20 TRiP line: y, sc, v; P{TRiP.HMS01553}attP2/TM3, Sb; the somatic daughterless driver: w; P{w+, GMR12B08-GAL4}attP2; the germline triple driver: P{otu-GAL4::VP16.1}; P{GAL4-Nos.NGT}40; P{GAL4::VP16-Nos.UTR}MVD1; and the stocks used for IPC ablation: w; P{w+, dilp2::GS-GAL4}/Cyo, and w; P{w+, UAS::Reaper}. During this work, wMel was introduced into the somatic daughterless driver, the germline triple driver, and the dilp2::GS-GAL4 driver, and the infected versions of these stocks were crossed to the TRiP or UAS:Reaper responders. DrosDel isogenic flies carrying wMel were used for real-time quantitative PCR analyses [122].

Food preparation and administration

The standard food recipe used was based upon that of the Bloomington Drosophila Stock Center [127]. The food was prepared in large batches that consisted of 20L water, 337g yeast, 190g soy flour, 1325g yellow corn meal, 96g agar, 1.5L Karo light corn syrup and 94mL propionic acid. To create yeast paste for this study, live bakers yeast was mixed together with water to create a smooth, thick paste. To create the “control food” used in this study, we mixed together 1.5mL ddH2O and 3.5mL of melted standard food in a narrow-mouthed vial, then let cool in an ice bucket to solidify the food suspension. The same procedure applied to creation of all other nutrient-altered foods used in this study. For “corn-syrup-enriched” food condition, 1.5mL Karo light corn syrup was used. For “yeast-enriched” food condition, 1.5mL of heat-killed yeast paste was used. The “BSA-enriched” food carried 0.4g BSA, 1.5mL water, and 3.5mL standard food. For the “sucrose-enriched”, “glucose-enriched” and “fructose-enriched” foods, fresh sugar solutions were prepared at a final concentration of 1g/mL, then 1.5mL of this concentrate was combined with 3.5mL standard food for each vial. The “glucose + fructose enriched” condition carried 0.75mL of 1g/mL glucose, 0.75mL 1g/mL fructose, and 3.5mL standard food. Alternate methods were used to prepare food for the other treatments. For the branched chain amino acid condition, the control condition contained 400μL water and 50μL DMSO mixed with 4.5mL standard food, whereas the experimental condition carried 200μL of 1mg/mL Arginine, 200uL of 1mg/mL Isoleucine and 50μL DMSO mixed with 4.5mL standard food. For the TORC1 testing, 50μL of either control DMSO or 30mM rapamycin/DMSO stock was mixed into 5mL standard food. For tests of IPC function, 50μL of either control DMSO or a 10mM mifepristone-DMSO stock was mixed into 5mL standard food.

Laboratory Drosophila stocks were maintained on standard food at 23–24°C. Identical population density was used in all vials, and control and experimental conditions run in parallel. Flies of the genotype w; Sp/Cyo; Sb/TM6B were used in all imaging experiments that assessed nutrition as the only variable. In the cases where crosses were needed to drive expression from TRIP line stocks or the dilp2:GAL4 stocks were used, we performed all crosses using identical population density and female age distribution in all vials, with control crosses always run in parallel. Virgin female flies were collected during the first 3 days of eclosion only, then subjected to nutrient conditions. The procedure was to collect a range of 0–24 hour old adults, age these young flies for 2 days on standard food, and expose to treatment conditions for 3 more days. The mixture of D. melanogaster and D. simulans flies collected from nature likely varied in age. These flies were also exposed to standard food for 2 days, and transferred to experimental food for 3 days. In the case of IPC ablation, the collected flies were allowed to mature 2 days, then transferred to mifepristone-containing food or DMSO control food. The flies were maintained on this food for 14 days, transferring the population to a fresh vial every 3 days of the treatment period. After this was completed, the flies were exposed to nutrient-altered food for 3 days.

Tissue staining, imaging, and analysis

Samples were prepared from a minimum of 10–15 flies per condition in each replicate. Ovary dissection, fixation, and propidium iodide staining were done as previously described in order to label germline Wolbachia nucleoids [38]. Ovarian tissues for all samples in each replicate were mounted on slides in parallel to ensure maximal consistency in sample compression between slide and coverslip. All samples were then imaged on a Leica SP2 confocal microscope at 63X magnification with 1.5X zoom. Experimental samples verified to exhibit the same degree of compression as the control sample were pursued further, while any experimental samples deviating from that were discarded. Z-series images were acquired from each egg chamber of interest at 1.5 μm intervals. Uniform intensity settings were applied to all egg chambers imaged within each replicate. A minimum of 7–10 oocytes were ultimately imaged from each condition, with all experimental oocytes matched for morphological consistency against control oocytes of the same replicate. Using this rigorous method, significant fold-differences in Wolbachia titer were consistently identified between control and experimental conditions, regardless of the baseline quantity of Wolbachia detected in each replicate.

To quantify Wolbachia titer in the confocal images, we used established methods to identify the deepest possible focal plane where Wolbachia are clearly visible in all samples tested for each replicate [32]. The images were processed in Photoshop to remove everything from the images except oocyte Wolbachia, which were then quantified using the Analyze Particles feature in Image J. This analysis ultimately quantifies the Wolbachia nucleoids carried per oocyte, or per nurse cell, within a single, representative focal plane of each egg chamber. Although the graphical data displayed in the figures present all experimental averages as normalized against the control averages, all statistical calculations were run by comparing each condition only against controls that were run in parallel. Significant differences were indicated by ANOVA. A minimum of 2–3 replicates were performed for most germline staining experiments described in this study. The only exception was the experiment in which Wolbachia titer responses were analyzed in both brain and ovary tissues. In that case, single replicates were done for each type of tissue stained, with all conditions run in parallel.

To analyze Wolbachia titer by real-time quantitative PCR, single flies were homogenized with a pestle in 250 μl of Tris HCl 0.1M, EDTA 0.1M and SDS 1% (pH 9) and incubated for 30 minutes at 70 ºC. After 35 μl of KAc were added the sample was incubated 30 minutes on ice, centrifuged for 15 minutes at 13.000 rpm at 4ºC and the supernatant stored. Samples were diluted 100x for qPCR. qPCr was performed as described previously [122], using the CFX384 Real-Time PCR Detection System and iQ SYBR Green Supermix (both BioRad). The relative amount of Wolbachia was calculated with the Pfaffl method [128], using the primers for the gene wsp to determine Wolbachia DNA levels and primers for host Rpl32 and Actin5C genes to normalize male and female samples, respectively [122]. Data from males were analyzed using a linear model on the log of the relative wsp levels (Im in R) [129]. Data from females were analyzed using a mixed linear model on the logs of relative wsp levels (lmer in R).

To analyze Wolbachia in the Drosophila central nervous system, brains were dissected and fixed as previously described [101]. Brains were incubated in anti-rabbit wsp antibody + PBST (0.1% Triton X-100) for 4 hours at room temperature or at least 12 hours at 4 degrees. For secondary antibody staining, goat anti-rabbit Alexa Fluor 546 (Invitrogen) was used at room temperature or at least 12 hours at four degrees. Actin labeling was done with phalloidin conjugated to Alexa 488, diluted 1:100 in PBST, for one hour at room temperature. Brain tissues were imaged on a Leica SP2 confocal microscope at 63X magnification. Brains were quantified with Leica LAF AS software. One representative focal plane per brain was scored. Cells containing one or more Wolbachia were scored as infected. Wolbachia aggregates larger than 10 microns² were scored as a “cluster” [101].

To assess Wolbachia nucleoid shape, we acquired Z-series images of stage 10A oocytes at 63X magnification with 5X zoom. Then we created a projection of 4 images from each Z-series, located just beneath the follicle cell layer, and measured the length of individual nucleoids using the “line” tool located within the Profile function of Quantification Tools in the Leica SP2 software. Elongation index was calculated as a function of length divided by width. It is assumed that the bacteria are random in orientation, and thus detecting a range of nucleoid morphologies ranging from spherical to rod-shaped is possible. Chi square tests were used to compare Wolbachia length and elongation index exhibited by bacterial populations from each treatment condition.

Supporting Information

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