Wolbachia enhances ovarian development in the rice planthopper Laodelphax striatellus through elevated energy production

Domain: biology
Published: 2025-12-13
Editorial outcome: minor_revision

Abstract
The endosymbiont Wolbachia can both benefit host nutrition and manipulate host reproduction to its own advantage. However, the mechanisms of its nutritional benefits remain unclear. We show that Wolbachia enhances ovarian development in the small brown planthopper Laodelphax striatellus by boosting energy production. Wolbachia-infected females have increased fecundity, accelerated ovarian development, and prolonged oviposition. Enhanced activity of mitochondrial complex I is linked to increased ATP production and the expression of energy metabolism-related genes. We further identify that Wolbachia-synthesized riboflavin is crucial for ATP production and ovarian development. A riboflavin transporter, slc52a3a, positively correlates with Wolbachia density and is required for normal ovarian maturation. Our findings demonstrate that Wolbachia-produced riboflavin drives energy production and accelerates ovarian maturation, thus improving host fecundity. This research reveals insights into symbiont-host metabolic interactions and underscores the role of nutrient delivery in symbiosis.

Introduction
The symbiotic relationship between insects and bacteria often exhibits a dual nature, wherein symbiotic bacteria can simultaneously benefit and harm their hosts, leading to their characterization as “Jekyll and Hyde” symbionts1–4. This duality highlights the complex and dynamic interactions between symbionts and their hosts. Wolbachia is one of the most notable examples. Although Wolbachia is known for its harmful reproductive manipulations, such as inducing male killing and cytoplasmic incompatibility (CI) that leads to offspring mortality5,6, recent studies have also revealed beneficial effects of Wolbachia on host fecundity in some insects7,8. For instance, Wolbachia has been shown to increase host fecundity under conditions of stress or nutrient deprivation and can be essential for oogenesis9–12. Despite these insights, the molecular mechanisms by which Wolbachia enhances host fecundity are not entirely clear.

Among the various mechanisms that promote fecundity, the role of Wolbachia in providing vitamin B to their hosts has attracted increasing interest. In the bed bug Cimex lectularius, Wolbachia synthesize biotin (vitamin B7) and riboflavin (vitamin B2), which are essential for host growth and fecundity13,14. Comparative genomic studies show that the riboflavin synthesis pathway is highly conserved among different Wolbachia strains, underscoring its importance in host-symbiont interactions15. Riboflavin is a water-soluble molecule that plays a critical role in energy metabolism and cellular function. It is a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential cofactors for mitochondrial electron transport chain enzymes, and thereby supports energy production and oxidative phosphorylation16,17. However, little is known about the effects of riboflavin synthesized by Wolbachia on host energy function and reproduction.

Wolbachia induces complete CI while simultaneously enhances female fecundity in the small brown planthopper (Laodelphax striatellus, SBPH), a major rice pest that feeds exclusively on plant sap18,19. This dual functionality allows Wolbachia to establish and rapidly spread within SBPH populations, as evidenced by its near 100% infection rate in field surveys20,21. Our previous work has shown that Wolbachia-provided biotin and riboflavin play significant role in promoting SBPH fecundity14. In this study, we combine transcriptomic analyses and experimental approaches to demonstrate the mechanisms by which riboflavin enhances reproductive capacity. We find that Wolbachia infection increases energy production in early adult SBPH, accelerating ovarian development and enhancing female fecundity. We further reveal that this effect is mediated by Wolbachia-synthesized riboflavin and the host’s riboflavin transporter, slc52a3a. These findings provide a clear understanding of the molecular and cellular mechanisms underlying Wolbachia’s reproductive contributions.

Results
Wolbachia enhances female ovarian development in SBPH
We first compared the biological performance of Wolbachia-infected (WI) and Wolbachia-uninfected (WU) SBPH (Fig. 1). Wolbachia infection significantly increased female fecundity by 53% (Fig. 1a). In addition, the hatch rate of deposited eggs was significantly higher in WI lines (Fig. 1b). After hatching, WI and WU planthoppers took similar times to develop from nymphs to adults. However, WI nymphs exhibited a significantly higher emergence rate (Supplementary Fig. 1), and the emerged adults lived much longer than the WU adults (Fig. 1c). We did not observe any significant differences in adult sex ratio or wing length variance (Supplementary Fig. 1).

To get more details on how Wolbachia affects female egg production, we counted the number of eggs laid by a female every two days until they died (Fig. 1d). WI females laid significantly more eggs in the first few days after emergence compared to WU females. They also had a longer lifespan and extended egg-laying period. Furthermore, during the first six days after emergence, WI females started laying eggs earlier than WU females, and the numbers of eggs laid per day were significantly higher (Fig. 1e). The number of mature oocytes was also significantly higher in WI ovaries than in WU ovaries on the third and fifth days after adult emergence (Fig. 1f). This suggests that WI ovaries develop earlier and faster than WU ovaries. We also found that WI ovaries grew faster and larger than WU ovaries (Fig. 1g).

Wolbachia upregulates gene expression of mitochondrion-related pathways in female SBPH
To explore the host genes regulated by Wolbachia in female SBPH, we performed RNA sequencing on the whole bodies of WI and WU third instar nymphs (N3), fifth instar nymphs (N5), and 1-day-old (A1dF), 3-day-old (A3dF), and 5-day-old (A5dF) adult females after emergence (Fig. 2a). After mapping the RNA-seq reads to the chromosome-level genome of SBPH22, we used the WGCNA analysis to classify SBPH genes into 26 different gene expression modules (each assigned a different color) depending on infection status, life stage and developmental time (Supplementary Fig. 2 and Supplementary Data 2). The 256 genes belonging to magenta and grey60 modules were screened out as their expression were mostly significant by both Wolbachia infection status and the difference between nymphs and adults. GO (Gene Ontology) enrichment analysis of these genes yielded terms related to mitochondrial outer membrane translocase complex pathway, nuclear outer membrane-endoplasmic reticulum membrane network pathway, intracellular and endoplasmic reticulum protein-containing complex pathway and endopeptidase complex pathway (Fig. 2d and Supplementary Data 2). Therefore, we hypothesize that mitochondria are involved in the physiological regulation of planthoppers by Wolbachia.

Wolbachia were found to have a higher density in the midgut and Malpighian tubules (Fig. 2b, c). Because of this and the finding that the ovaries of WI planthoppers develop faster (Fig. 1f, g), we dissected all these tissues and did RNA-seq to analyze the differential expression of planthopper genes in these tissues (Fig. 2c). We found that the number of differentially expressed genes (DEGs) in the ovaries of WI and WU females were significantly higher than those in the midgut and Malpighian tubules (Supplementary Fig. 2). Interestingly, in the GO analysis, mitochondrial outer membrane protein complexes were consistently enriched in the DEGs of multiple tissues in WI and WU females (Fig. 2d). Several pathways associated with ovarian development and energy supply like those in the ovarian fusome organization and organic acid metabolism process were significantly enriched in WI females (Supplementary Fig. 3). During the developmental stages of the females, many genes related to energy metabolism showed a significant upward trend, and among them, the genes related to energy production were significantly upregulated (Fig. 2d and Supplementary Data 3). In addition, many genes associated with energy metabolism pathways (such as carbohydrate metabolism, insect hormone biosynthesis, and insulin and mTOR signaling pathway) were highly expressed in WI females (Supplementary Data 3). These results indicated that Wolbachia may upregulate energy production systematically in female SBPH.

Wolbachia increases ovarian development by improving the energy production
Although energy production-related genes were upregulated in WI female SBPH, the expression of mitochondrial genome-encoded genes and mitochondrial density were similar between WI and WU SBPH, both in the body and ovaries (Supplementary Fig. 4a, b). We found that SBPH nuclear genes mainly associated with complexes I and II of the mitochondrial respiratory chains exhibited differential expression (Supplementary Fig. 4c–f). The involvement of mitochondrial complexes in oxidative phosphorylation is the primary source of energy production (Fig. 3a). Further analysis using Kit-based assays revealed that the mitochondrial complex I activity, rather than complex II activity, was significantly higher in 1-day-old adult WI females (Fig. 3b, c). This may explain the significantly higher ATP content in the body of 1-day-old WI females and ovaries of 1-day-old and 3-day-old WI females (Fig. 3d). However, this difference diminished later (Fig. 3d), implying a more rapid energy expenditure at the beginning.

To verify the effect of energy on ovarian development, we fed fifth instar Wolbachia-infected females with rotenone, a mitochondrial complex I inhibitor which affects energy production (Fig. 3e). Feeding of 5 μM rotenone significantly reduced energy production without affecting SBPH survival or Wolbachia density (Supplementary Fig. 5). The ATP content was significantly higher than that of the control group at the 1 and 5 days after treatment (Fig. 3f). The number of mature oocytes in the ovaries of 3-day-old rotenone-fed SBPH females was significantly lower than that of the control group (Fig. 3g), and the oocytes developed more slowly after rotenone treatment (Fig. 3h), suggesting that the energy is essential for normal ovarian development. Additionally, we knocked down the gene expression of ATPeF1B, a gene important in ATP synthesis (Fig. 3i), and found that when ATP content decreased in 1-day-old females (Fig. 3j), both the ovarian development and the number of mature oocytes in WI planthoppers were significantly reduced (Fig. 3k, l). These results highlight the importance of energy production, particularly mitochondrial complex I, on ovarian development.

Wolbachia-synthesized riboflavin promotes energy production
We previously reported that the genome of SBPH’s Wolbachia (the wStri strain) encodes a complete set of riboflavin synthesis genes14. The riboflavin content in WI female adults was higher than the WU females, especially significantly higher in 1-day-old females (Fig. 4a). When both WI and WU SBPH were maintained on a riboflavin-deficient diet, WU females exhibited higher mortality than WI females. However, when both groups were provided with a riboflavin-supplemented diet, the survival rate of WU SBPH became comparable to that of WI individuals (Supplementary Fig. 6). Riboflavin plays an important role in energy production, serving as essential cofactors for mitochondrial complex I and complex II in the forms of FAD and FMN, respectively (Fig. 3a). To verify whether riboflavin synthesized by Wolbachia promotes energy production, we transferred wStri Wolbachia to a Spodoptera frugiperda sf9 cell line (Fig. 4b) and measured the riboflavin contents in both cell lines. The riboflavin content in the wStri-transfected sf9 cells was significantly higher than that of the untransfected sf9 cells (Fig. 4c), and the ATP content was eight times that of the control group (Fig. 4d). These results indicate that Wolbachia-synthesized riboflavin is a key factor in enhancing energy production.

We then added riboflavin to the artificial diet of WU fifth-instar nymphs and measured the ATP content and the ovarian development of the emerged female adults (Fig. 4e). Nymphs fed with riboflavin containing diet had higher emergence rate (Fig. 4f), while the ATP content in the newly emerged females was significantly higher (Fig. 4g). On the third day after emergence, mature oocytes were observed in the ovaries of both groups of females fed with riboflavin, while nearly no mature oocytes were found in the ovaries of unfed females (Fig. 4h, i). However, when riboflavin was provided to WI planthoppers, this beneficial effect was no longer observed (Supplementary Fig. 7), suggesting that riboflavin is more critical for WU planthoppers. Consequently, the increase in riboflavin content enhances the energy production and ovary development of WU female planthoppers.

Wolbachia-upregulated riboflavin transporter slc52a3a is required for ovarian development
Riboflavin was transported across cell membranes by the SLC52A family proteins23. We identified a SLC52A member, the slc52a3a gene (GenBank: RZF44203), in the SBPH genome and found SLC52A3A is conserved across insect orders (Supplementary Fig. 8). slc52a3a was upregulated in WI nymphs and early female adults compared to WU SBPH (Fig. 5a). Like Wolbachia, slc52a3a was highly expressed in the midgut and Malpighian tubules and peaked on days 9 and 11 in female adults (Supplementary Fig. 9). Wolbachia density was found to be positively correlated with slc52a3a expression in various developmental stages and adult tissues (Fig. 5b). Injecting WI 5th nymphs with dsRNA targeting slc52a3a significantly decreased the emergence rates, and many planthoppers died during emergence and molting (Fig. 5c–e). The females that emerged showed higher mortality, less mitochondrial complex I activity, less ATP content, and fewer mature oocytes compared to females that were not treated in the nymph stage (Fig. 5f–j). These results indicate that the promoting effect of riboflavin on host ovarian development requires slc52a3a. However, when slc52a3a was silenced in 1-day and 5-day old female adults, no significant effect was observed (Fig. S10), including 9-day and 11-day old SBPH, where slc52a3a showed an expression peak. This indicated that riboflavin’s effect on ovarian development occurs mainly in the early post-emergence period.

Methods
Insect rearing
The Laodelphax striatellus SBPH planthoppers were collected in rice paddies in Nanjing. Planthoppers were reared on rice seedlings and maintained in an environment chamber at 25°C ± 1°C, 60% relative humidity and a 16L:8D photoperiod. The population was treated with 1 mg/ml tetracycline to obtain WU planthoppers. To produce WI planthoppers, as well as to eliminate the effect of factors unrelated to Wolbachia on SBPH, Wolbachia-containing haemolymph was injected into nymphs of WU planthoppers as previously described14. Approximately once a month, Wolbachia infection status of the planthopper populations was checked by PCR amplification of the Wolbachia surface protein (wsp) gene (Supplementary Data 4).

Biological performance assay
Fifth-instar nymphs were collected and reared in glass tubes containing several rice seedlings until emergence. The emergence rate, sex ratio, wing length type of the emerged adults, and the lifespan of female adults were recorded. Newly emerged female adults were paired with two male adults for mating. The number of eggs laid by the female, the number of hatched eggs and the times from hatching to emergence were recorded. To investigate ovarian development in female adults, ovarian samples from newly emerged individuals were dissected under a stereomicroscope. Images were captured using a stereo fluorescence microscope SMZ25 equipped with the NIS-Elements BR 4.50.00 digital imaging system (Nikon, Tokyo, Japan).

Quantification of Wolbachia densities
The density of Wolbachia was determined using quantitative PCR (qPCR) on DNA samples extracted from SBPH as previously described14. Total DNA was isolated using the FastPure Blood/Cell/Tissue/Bacteria DNA Isolation Mini Kit (Vazyme Biotech, Nanjing, Jiangsu, China). The copy numbers of the Wolbachia wsp (AF020080) and the SBPH single-copy gene actin (AY192151) were quantified by qPCR using ChamQ SYBR qPCR Master Mix (Vazyme Biotech) on a QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The relative Wolbachia density was calculated as the ratio of wsp to actin copy numbers. Each DNA sample was analyzed in duplicate technical replicates. The primer sequences are provided in Supplementary Data 4.

Fluorescence in situ hybridization
The localization of Wolbachia in SBPH tissues was visualized using fluorescence in situ hybridization (FISH). Two rhodamine 5′-end labeled 16S rRNA Wolbachia-specific probes (W1: 5′-AATCCGGCC-GARCCGACCC-3′ and W2: 5′-CTTCTGTGAGTACCGTCATTATC-3′) were used for Wolbachia detection, while DAPI was employed to stain DNA. Midgut, Malpighian tubules, and ovary tissues from female SBPH were dissected and immediately fixed in 4% paraformaldehyde for 30 min. The samples were then processed according to established protocols25 and imaged using a Leica TCS SP8 laser confocal microscope (Leica Wetzlar, Germany).

Quantitative reverse transcription PCR (RT-qPCR)
Total RNA was extracted from the samples using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and reverse-transcribed into cDNA using HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech). Gene expression levels were quantified by qPCR, as described above. Each sample included two technical replicates. Relative gene expression levels were calculated using the delta Ct method, with the rps5 gene (XM_039430651) as a reference control. Primer sequences are listed in Supplementary Data 4.

Transcriptome sequencing and analysis
For the RNA-seq analyses, WI, WU nymphs in the 3rd-instar and 5th-instar and female adults of 1-day-old, 3-day-old, and 5-day-old were collected (Fig. 2A), with three biological replicates of at least 3 individuals for each stage. Additionally, the ovaries of 1-day-old females were dissected, as well as the midgut and Malpighian tubules of 3-day-old females, with three biological replicates of at least 10 individuals for each tissue. Total RNA was extracted using TRIzol. cDNA libraries were then prepared and sequenced on a BGISEQ-500 (BGI, Shenzhen, Guangdong, China) by Beijing Genomics Institution.

The sequenced reads were cleaned using SOAPnuke v.1.5.226 and fastp v.0.20.027. The clean reads were then mapped to the L. striatellus transcriptome (GCF_014465815.1)22 using STAR RNA-seq aligner v.2.7.5c28. The number of mapped reads was counted using the “–quantMode GeneCounts” parameter in STAR.

To identify differentially expressed genes (DEGs) between WI and WU samples from various tissues, gene expression was assessed using read counts with DESeq229 on R 4.4.230. Gene expression levels were measured as Fragments per Kilobase Million (FPKM). Genes with an average FPKM greater than 5 in either treatment were considered expressed. DEGs were identified based on the criteria: average FPKM > 5, log2(fold change of FPKM) > 0.585, and adjusted p-value < 0.05. Transcriptomic data from WI and WU whole insects were subjected to co-expression analysis using WGCNA and hierarchical clustering based on gene connectivity dissimilarity (1-TOM)31. Gene Ontology (GO) enrichment analysis was conducted using the R package “clusterProfiler”32.

ATP content, complex I and II activities
To measure ATP content in female SBPH, samples were homogenized in lysis buffer and centrifuged at 12,000 × g for 10 min to obtain the supernatant. ATP levels were quantified using an ATP assay kit (Beyotime Biotechnology, Shanghai, China) following the manufacturer’s instructions.

To assess the activities of mitochondrial complexes I and II, SBPH samples were homogenized in extraction buffer on ice. The homogenate was centrifuged at 600 × g for 10 min at 4°C to obtain the supernatant, which was further centrifuged at 11,000 × g for 15 min at 4°C to yield the final supernatant for activity assays. The activities of complex I and complex II were evaluated using micro mitochondrial respiratory chain activity kits for complex I and II (Solarbio, Beijing, China).

ATP content and complex activities were measured using a SpectraMax M5 microplate reader (Molecular Devices, San Jose, CA, USA) by detecting chemiluminescence and absorbance at specific wavelengths, respectively. To minimize variability caused by differences in protein concentration during sample preparation, samples were normalized to the same protein concentration using a BCA Protein Assay Kit (Sangon Biotech, Shanghai, China). Each treatment included at least three biological replicates, with three individuals per replicate.

Riboflavin content analysis
Riboflavin content was measured as previously described33. Female SBPH samples were homogenized in citrate buffer (pH=4.5) and enzymatically digested with Taka-diastase and acid phosphatase (Sigma-Aldrich, St. Louis, MO, USA) under dark incubation (37°C, 1 h). The hydrolysate was heat-treated (95°C, 30 min) with periodic agitation, followed by rapid cooling and centrifugation (800 × g, 5 min). The supernatant was inoculated into riboflavin-dependent Lactobacillus rhamnosus (ATCC, 7469) cultured in Difco™ Riboflavin Assay Medium (BD, Franklin Lakes, NJ, USA). After dark incubation (37°C, 19–24 h), absorbance at 630 nm was measured using the SpectraMax M5 microplate reader. Riboflavin concentrations were determined against a standard curve and normalized to sample weight to correct sampling bias.

Mitochondrial complex I inhibition by rotenone
The mitochondrial complex I inhibitor rotenone (Macklin Biochemical, Shanghai, China) was dissolved in acetone to prepare a stock solution with a concentration of 1 mg/ml. Rotenone was then added to the artificial diet34 at final concentrations of 0, 5, 10, and 25 μM. Fifth-instar nymphs were fed the treated diets until emergence. The survival rate, ATP content in female adults and Wolbachia density were measured as described above.

Riboflavin supplement experiment
To examine the impact of riboflavin on SBPH, the WI and WU SBPH were fed three different diets: a normal artificial diet, an artificial diet deficient in riboflavin, and a sucrose solution serving as a control. Their survival rates were recorded over a period of 15 days. In further experiments, fifth-instar nymphs were fed artificial diets supplemented with varying concentrations of riboflavin. The experiments were conducted in a rearing chamber under dark conditions. The emergence rates of WU nymphs, the ATP content in 1-day-old female adults and the number of mature oocytes in 3-day-old female adults were recorded.

Transfection of Wolbachia in cell lines
Freshly laid WI eggs of SBPH (as donors) were carefully collected and immersed in 1× phosphate-buffered saline (PBS). Following the method described by Li et al.35, the Wolbachia was co-cultured with Wolbachia-free Spodoptera frugiperda Sf9 cells. Once the Sf9 cell line was successfully infected with wStri, subsequent generations of cells were subjected for PCR confirmation to verify the presence of the wStri. This process was repeated until a stably wStri-infected Sf9 cell line was established. To measure the ATP and riboflavin contents, equal number of cells were sonicated after being selected out using a Countess II FL Automated Cell Counter (Thermo Fisher Scientific).

RNA interference (RNAi)
The slc52a3a gene was amplified from SBPH cDNA and cloned into the pClone007 vector (Tsingke, Beijing, China). The recombinant vector served as a template for PCR amplification, and the resulting product was used to synthesize double-stranded RNA (dsRNA) with the T7 High-Yield RNA Transcription Kit (Vazyme). The dsRNA was purified following a previously described method36 and adjusted to a final concentration of 5000 ng/μl. Fifth-instar nymphs or newly emerged female adults were microinjected with 250 ng of dsRNA using a Nanoliter 2010 Microinjection Pump (WPI, Sarasota, FL, USA). After the injected nymphs developed into adults, insects were collected for analyzing RNAi efficiency with RT-qPCR. The red fluorescent protein (RFP) gene was used as a control. The primer sequences are listed in Supplementary Data 4.

Statistical analysis
Ecological data and qPCR results were first tested for normality using the Shapiro–Wilk test, followed by Student’s two-sided t test and ANOVA with the Tukey post-hoc test for parametric analysis or the Wilcoxon rank sum test and Kruskal–Wallis test with Bonferroni correction for non-parametric analysis. Multiple comparisons were analyzed with the “agricolae” package37. The effects of Wolbachia infection and time on egg numbers and oocyte numbers were analyzed using a generalized linear model (GLM). Survival curves were compared using the Cox proportional hazards model. Fisher’s exact test was used for analyzing emergence rate, wing form ratio, and sex ratio. All statistical analyses were performed in R 4.4.230. The schematic illustrations were draw by Inkscape 0.92 (https://inkscape.org/).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This research reveals some new insights into symbiont-host metabolic interactions and underscores the role of nutrient delivery in symbiosis. In this study, Wolbachia infection elevated riboflavin levels, upregulated the expression of the riboflavin transporter slc52a3a, and enhanced the respiratory activity of mitochondrial complex I post-emergence, leading to increased ATP production. This rise in ATP content significantly accelerated ovarian development, enabling WI females to enter the oviposition phase earlier and lay more eggs. Nevertheless, this study is preliminary and lacks enough evidences. The main conclusion that Wolbachia enhances ovarian development by improving mitochondrial functions is not well supported by the data. Please see detailed major and minor comments below.\nMajor comments:\n1. Line 119: How to explain a reproductive manipulator Wolbachia were found to have a higher density in the midgut and\nMalpighian tubules, but not higher density in the ovarian?\n2. Line 154-156 and 174-176: “At emergence, ATP contents were significantly higher in WI females. However, this difference\ndiminished later, and by the fifth day post-emergence, ATP levels were lower in WI individuals” and “In 1-day-old adult\nfemales, NADH dehydrogenase activity of complex I was significantly higher in WI planthoppers (Fig. 3f), but the difference\nof succinate dehydrogenase activity in complex II was not significant (Fig. 3g)”\nHowever, as shown in Fig. 1, Wolbachia promotes fitness and ovary development in female SBPH, at least during the first\nsix days after emergence.\nHow can the inconsistencies between ATP levels, mitochondrial complex I activities, and biological phenotypes be\nexplained?\n3. Line 168-170 and 199-201: “No significant differences were observed in mitochondrial density and expression levels of\nmitochondria-encoded genes between WI and WU planthoppers in either the body or the ovaries (Fig. S6a, b)” and\n“Riboflavin plays an important role in mitochondrial function, serving as essential cofactors for mitochondrial complex I and\ncomplex II in the forms of FAD and FMN, respectively (Fig. 3e). There is currently a lack of direct evidence to support the\nnotion that Wolbachia enhances ovarian development through the improvement of mitochondrial functions.\n4. Line 236-237 and 243-245: “As in Wolbachia, slc52a3a was highly expressed peaked on days 9 and 11 in female\nadult(Fig. S9b)” However, when slc52a3a was silenced in female adults, no significant effect is observed (Fig S11),\nindicating that riboflavin’s effect on ovarian development occurs mainly in the early post-emergence period. What is the\nfunction of slc52a3a in female adult? Why as in Wolbachia, slc52a3a was highly expressed peaked on days 9 and 11 in\nfemale adult?\n5. As shown in Fig. 5: “Wolbachia upregulates the expression of SBPH’s riboflavin transporter slc52a3a to promote ovary\ndevelopment”\nDoes slc52a3a influence mitochondrial function, mitochondrial complex activity, or ATP levels? If not, what is the\nrelationship between slc52a3a and a reproductive manipulator that enhances insect ovarian development through elevated\nmitochondrial activity?\n6. The abundance of Wolbachia is lowest in the ovary than other tissues. How is riboflavin produced by Wolbachia in other\ntissues transported to the ovary?\n7. There are many genes highly expressed in mitochondria of WI (such as MDH2, MQO, OGDH, ATPeV0A, SLC25A1,\nMSS51, TOM20). Why just choose the gene slc52a3a for the study?\nMinor comments:\n1. Line 1-3: The title should be presented specifically for the system used in this study. It is more beneficial for readers to\nunderstand the article when the symbiotic bacteria Wolbachia and the small brown planthopper Laodelphax striatellus are\nclearly mentioned in the title.\n2. Line 86-87: “We also found that WI ovaries grew faster and larger than WI ovaries (Fig. 1g).” not correctly stated.\n3. Line 158 and 413: “Mitochondrial inhibition by rotenone” not correctly stated. Rotenone serves as a mitochondrial complex\nI inhibitor. There is a difference between mitochondrial function and mitochondrial complex I function.\n4. Line 161-164 and Line 203-205: The two instances of Fig 4c and 4d present distinct content, and there is no Fig 3c and 3d\nin the result section.\n5. Line 272: There are no known cellular mechanisms underlying Wolbachia's role in host reproduction in this process.\n6. Line 42: “recent studies have also revealed subtle yet significant beneficial effects on host fecundity”. The sentence\ndoesn’t make sense.\n7. Fig. 1c, 1d, 1e, 1f: Significance analysis is for a difference at all time points?", "reviewer_2": "Wolbachia are common endosymbionts that have profound effects on the reproduction of their insect hosts, with some\nstrains inducing cytoplasmic incompatibility, feminization, parthenogenesis and male-killing, while others can also provide\ndirect costs or benefits to reproduction. In this study, the authors investigate the mechanisms for enhanced female fertility in\nthe planthopper Laodelphax striatellus induced by Wolbachia. Two of their central conclusions are that 1. Wolbachia\nincreases female fertility by increasing mitochondrial activity and that 2. that the riboflavin produced by Wolbachia is\nresponsible for the increase in female fertility. While some of the findings provide important insights I am not fully convinced\nby these main conclusions of the study. Conclusion 1 is based on the data from Figures 2 (showing that expression of genes\nrelating to mitochondrial function are upregulated in the Wolbachia-infected WI line) and 3 (showing that mitochondrial\nfunction as measured by ATP content is improved). While the differences in gene expression are clear, I am not convinced\nby a direct link between Wolbachia and mitochondrial activity which is based on a single time point with low replication,\nrather than there being a clear overall pattern. For conclusion 2, the same issue arises and the experiments\nmanipulating riboflavin content only involve the uninfected line and not the WI line. I do not have any major issues with the rest of the\nmanuscript and the limitations raised here could be addressed by completing a full set of treatments with greater replication.\nThe current level of replication for these experiments should also be justified, especially when other experiments have n\nvalues far higher than 3-4. Please see more detailed comments on these issues below.\nLine 40 – In what way is CI harmful to the host that carries Wolbachia? CI can be beneficial by giving a relative advantage to\nfemales that are infected.\nLine 42-45 – The phrasing here is misleading because it implies that beneficial effects (either general or environment\nspecific) on fecundity are a general feature of Wolbachia strains. However this is only the case for some systems. In others\nsuch as mosquitoes, and especially in recent Wolbachia-host associations (such as deliberate transfers through\nmicroinjection) Wolbachia can have clear costs to fecundity. The authors should make it clear that effects of Wolbachia can\nbe extremely host and strain-specific and the insights of this study may not apply to other systems.\nFigure 1. Panels a, d and e all show egg counts- are these from three separate experiments done with different methods?\nThere is no information in the methods about how these comparisons differ.\nAlso for Figure 1, it would be useful to present individual data points on a scatter dot plot rather than means and SEMs. Bar\ncharts can be misleading because they obscure the distribution of the data.\nLine 87 – There is not enough data to claim that ovaries grew faster in the WI line as there is no quantification of this\nphenotype. Furthermore, the mature oocytes for WU look larger than WI at Day 5 in the images in Figure 1. Is this because\nthe scales are different? The image resolution is too low for me to be able to tell.\nLine 151 – The content of this section does not match the claim in this subheading. In Figure 3 the authors show higher ATP\ncontent in WI females compared to WU females early on, then the opposite pattern at day 5. There is no clear pattern of\nimproved ATP content in WI and overall these effects are not convincing due to a low level of biological replication (typically\nn = 4). The effects of rotenone also do not appear to be substantial and only the WI line is measured. In figure 3d there is no\nquantification of oocyte developmental rates (only single representative images). Finally, the authors do not find any clear\ndifferences between WI and WU in terms of mitochondrial energy production (Figures 3f-g and supplements), with the claims\nin line 177 being based only on a single time point for complex I with n = 3. Again, in these comparisons the authors should\npresent individual data points rather than means and SEM only. Overall I am not convinced from the experiments here of a\ndirect link between Wolbachia presence and mitochondrial function.\nLine 162 – The references to Fig 4c and 4d should be to figure 3.\nLine 197-199 – This claim is not supported- WU individuals performed worse than WI across all three diets- although the\ndifference on the diet containing riboflavin is not significant the WU line still has lower survival. To claim that Wolbachia-\nsynthesized riboflavin improves survival would require testing for an interaction between Wolbachia status and diet. Based\non the survival curves it looks like WU generally just has lower fitness rather than there being a specific effect of\nriboflavin.\nLine 203 and Figure 4 – It is clear from this experiment that riboflavin supplementation improves emergence/mt\nfunction/oocyte maturation in the WU line, but it is unclear why only the WU line was tested. If Wolbachia-synthesised\nriboflavin is important, then you would expect to see a more pronounced improvement in the WU line compared to WI."}, "author_response": "We thank the two reviewers for their thoughtful comments and suggestions. We have helped us greatly improve this manuscript. You will find below our point-by-point answer to each individual comment in blue. Given the extensive revisions we've made, we recommend reading the clean version provided for better readability.\nReviewer #1 (Remarks to the Author):\nThis research reveals some new insights into symbiont-host metabolic interactions\nand underscores the role of nutrient delivery in symbiosis. In this study, Wolbachia\ninfection elevated riboflavin levels, upregulated the expression of the riboflavin transporter slc52a3a, and enhanced the respiratory activity of mitochondrial complex I\npost-emergence, leading to increased ATP production. This rise in ATP content\nsignificantly accelerated ovarian development, enabling WI females to enter the\noviposition phase earlier and lay more eggs. Nevertheless, this study is preliminary\nand lacks enough evidences. The main conclusion that Wolbachia enhances ovarian\ndevelopment by improving mitochondrial functions is not well supported by the data.\nPlease see detailed major and minor comments below.\nResponse: We sincerely appreciate the reviewer’s insightful feedback and suggestions\nfor improving our manuscript. In response, we have added new data to support our\nclaims, as outlined below. Additionally, we have mortified our statements on how\nenergy production, rather than mitochondrial functions affects ovarian development\nand fecundity.\nMajor comments:\n1. Line 119: How to explain a reproductive manipulator Wolbachia were found to\nhave a higher density in the midgut and Malpighian tubules, but not higher density in\nthe ovarian?\nResponse: Our previous research showed that in rice planthoppers, Wolbachia enters\nthe ovary from the anterior end of the ovarian tubules during its development,\npromoting vertical transmission (Guo et al., 2018). Therefore, it is normal for the titer\nin the ovary will be low when the ovary is not yet fully developed.\nGuo, Y., Hoffmann, A. A., Xu, X.-Q., Mo, P.-W., Huang, H.-J., Gong, J.-T., . . .\nHong, X.-Y. 2018. Vertical transmission of Wolbachia is associated with host\nvitellogenin in Laodelphax striatellus. Frontiers in Microbiology, 9, 2016.\n2. Line 154-156 and 174-176: “At emergence, ATP contents were significantly higher\nin WI females. However, this difference diminished later, and by the fifth day\npost-emergence, ATP levels were lower in WI individuals” and “In 1-day-old adult\nfemales, NADH dehydrogenase activity of complex I was significantly higher in WI\nplanthoppers (Fig. 3f), but the difference of succinate dehydrogenase activity in\n1\ncomplex II was not significant (Fig. 3g)”\nHowever, as shown in Fig. 1, Wolbachia promotes fitness and ovary development in\nfemale SBPH, at least during the first six days after emergence.\nHow can the inconsistencies between ATP levels, mitochondrial complex I activities,\nand biological phenotypes be explained?\nResponse: We appreciate the reviewer’s insightful question regarding the apparent\ninconsistency between ATP levels, mitochondrial complex I activity, and the observed\nfitness/ovary development phenotype. To clarify, we measured ATP production in the\novaries of WI females during the early stages of adult development. The results show\nthat: (1) At 3 d post-emergence, ATP levels in the ovaries of WI females were\nsignificantly higher than those in WU females (Fig. 3d). This finding is consistent\nwith our observation that ovarian development in WI is significantly faster than in\nWU on day 3, supporting the conclusion that increased ATP production accelerates\novarian development. (2) By 5 d post-emergence, ATP levels in WI ovaries became\nslightly lower than those in WU ovaries, consistent with the whole-body ATP trend.\nThese data suggest that WI females require elevated ovarian ATP levels during early\nemergence (1–3 d) to support rapid ovary development, while the reduced ATP at 5 d\nmay reflect a stabilized developmental state. Thus, we believe that the early ATP\nsurge (rather than sustained high levels) appears critical for ovarian fitness.\n3. Line 168-170 and 199-201: “No significant differences were observed in\nmitochondrial density and expression levels of mitochondria-encoded genes between\nWI and WU planthoppers in either the body or the ovaries (Fig. S6a, b)” and\n“Riboflavin plays an important role in mitochondrial function, serving as essential\ncofactors for mitochondrial complex I and complex II in the forms of FAD and FMN,\nrespectively (Fig. 3e). There is currently a lack of direct evidence to support the\nnotion that Wolbachia enhances ovarian development through the improvement of\nmitochondrial functions.\nResponse: We have revised the text to clarify that Wolbachia infection elevates ATP\nlevels and upregulates genes related to energy production, while removing\nunsupported claims about enhanced mitochondrial function. This adjustment\nimproves clarity and aligns with the current experimental evidence.\n4. Line 236-237 and 243-245: “As in Wolbachia, slc52a3a was highly expressed\npeaked on days 9 and 11 in female adult (Fig. S9b)” However, when slc52a3a was\nsilenced in female adults, no significant effect is observed (Fig S11), indicating that\nriboflavin’s effect on ovarian development occurs mainly in the early post-emergence\nperiod. What is the function of slc52a3a in female adult? Why as in Wolbachia,\nslc52a3a was highly expressed peaked on days 9 and 11 in female adult?\nResponse: We are unsure why the expression level of slc52a3a increased at days 9\nand 11. This may correlate with a peak in Wolbachia titer at this stage, which could\ninfluence slc52a3a expression. However, when we injected dsslc52a3a into females 5\n2\ndays post-emergence and ensured RNAi efficiency on days 9 and 11, we observed no\nsignificant changes in ATP levels or egg-laying behavior between days 5 and 15 (Fig.\nS10). These findings suggest that, despite the peak expression of slc52a3a on days 9\nand 11, its function may not be as critical to female ovarian development during this\nperiod. Our study focused more on the role of riboflavin in ovarian development\nduring the early post-emergence period, specifically within the first few days of adult\nemergence. Therefore, we concentrated on the effect of slc52a3a gene on ovarian\ndevelopment during early post-emergence period.\n5. As shown in Fig. 5: “Wolbachia upregulates the expression of SBPH’s riboflavin transporter slc52a3a to promote ovary\ndevelopment”. Does slc52a3a influence mitochondrial function, mitochondrial complex activity, or ATP levels? If not, what is\nthe relationship between slc52a3a and a reproductive manipulator that enhances insect\novarian development through elevated mitochondrial activity?\nResponse: Yes, slc52a3a does influence mitochondrial function, mitochondrial\ncomplex activity, and ATP levels. We have added new data showing that silencing\nslc52a3a in fifth-instar nymphs significantly inhibited ATP production and complex I\nactivity in 1-day-old females (Fig. 5g, h). Additionally, we confirmed the suppression\nof ovarian development with corresponding images (Fig. 5j). However, when we\nexamined the effect of slc52a3a knockdown on reproductive regulators, such as Vg,\nwe found no significant impact on Vg expression (Figure below). This suggests that\nslc52a3a’s role in promoting ovarian development is likely independent of Vg\nregulation.\nFigure legend: The expression level of Vg gene in female SBPH following dsslc52a3a\ntreatment in fifth-instar nymphs. The difference between dsRFP and dsslc52a3a samples was\nstatistically analyzed using the Wilcoxon rank-sum test. ns, not significant.\n6. The abundance of Wolbachia is lowest in the ovary than other tissues. How is\nriboflavin produced by Wolbachia in other tissues transported to the ovary?\nResponse: This is an excellent comment. During our research, we attempted to\n3\ninvestigate how riboflavin is transported from other tissues to the ovary. However,\ndue to technological limitations, we were unable to conduct direct approaches like\nisotope labeling techniques. In response to the reviewer’s comments, we further\nexplored the distribution of riboflavin across tissues. Unfortunately, the small size of\nSBPH tissues limited our ability to achieve conclusive results. Therefore, we can only\nhypothesize that the transport of riboflavin to the ovary may be facilitated by the\nriboflavin transporter SLC52A3A (Fig. 5). We hope to conduct more in-depth\nresearch in the future to better address this question once we resolve this issue.\n7. There are many genes highly expressed in mitochondria of WI (such as MDH2,\nMQO, OGDH, ATPeV0A, SLC25A1, MSS51, TOM20). Why just choose the gene\nslc52a3a for the study?\nResponse: The slc52a3a gene was selected for study because it encodes a riboflavin\ntransporter protein, which could provide valuable insights into the role of riboflavin in\nenhancing the reproductive capacity of rice planthoppers influenced by Wolbachia.\nAdditionally, we also investigated the impact of knocking down the mqo gene (a\ncritical gene for complex II activity) on ovarian development in SBPH. However, the\nresults showed no significant effect on female ovarian development and survival\ncurves, although we observed some influence on emergence rate (Figure below).\nThese findings support the reviewer’s suggestion that mitochondrial genes may not\ndirectly contribute to the increased reproductive capacity of rice planthoppers. Given\nthat the results of mqo are not strongly related to the central theme of the paper, we\ndecided not to include them in the revised manuscript in order to maintain the clarity\nand flow of the narrative.\nFigure legend: Biological phenotype following RNAi of mqo in fifth-instar nymphs.\n(a) Relative expression levels of mqo normalized to rps5 in WI SBPH at 3 days post-dsRNA\ntreatment in newly emerged adult females. n = 10-12. (b) Emergence rate. n = 193-211. (c)\nSurvival curves of female adults. n = 50. (d) Number of mature oocytes. n = 6-12. Data are\npresented as mean ± SEM. Statistical analyses were performed using Student’s t test for panel\n(a), Chi-squared test for panel (b), the Cox model for panel (c), and Wilcoxon rank-sum test\nfor panel (d). Statistical significance levels are indicated by stars on the panels: *, P < 0.05;\n***, P < 0.001; ns, not significant.\n1. Line 1-3: The title should be presented specifically for the system used in this study.\nIt is more beneficial for readers to understand the article when the symbiotic bacteria\nWolbachia and the small brown planthopper Laodelphax striatellus are clearly\nmentioned in the title.\nResponse: We have revised the title accordingly.\n2. Line 86-87: “We also found that WI ovaries grew faster and larger than WI ovaries\n(Fig. 1g).” not correctly stated.\nResponse: We have corrected this text.\n3. Line 158 and 413: “Mitochondrial inhibition by rotenone” not correctly stated.\nRotenone serves as a mitochondrial complex I inhibitor. There is a difference between\nmitochondrial function and mitochondrial complex I function.\nResponse: We have revised the text.\n4. Line 161-164 and Line 203-205: The two instances of Fig 4c and 4d present\ndistinct content, and there is no Fig 3c and 3d in the result section.\nResponse: Thank you for the correction. The Fig 4c and 4d in lines 161–164 should\nbe Fig 3c and 3d. We have revised the text.\n5. Line 272: There are no known cellular mechanisms underlying Wolbachia's role in\nhost reproduction in this process.\nResponse: We have revised the text to be more cautious.\n6. Line 42: “recent studies have also revealed subtle yet significant beneficial effects\non host fecundity”. The sentence doesn’t make sense.\nResponse: We have revised the text.\n7. Fig. 1c, 1d, 1e, 1f: Significance analysis is for a difference at all time points?\nResponse: For Figures 1c, 1d, 1e, and 1f, the data were analyzed using a generalized\nlinear model (GLM) to assess the effects of Wolbachia infection and time on egg and\noocyte numbers. The significance analysis labeled on figures revealed a general\ndifference in the effect of Wolbachia.\nReviewer #2 (Remarks to the Author):\nWolbachia are common endosymbionts that have profound effects on the reproduction\nof their insect hosts, with some strains inducing cytoplasmic incompatibility,\nfeminization, parthenogenesis and male-killing, while others can also provide direct\ncosts or benefits to reproduction. In this study, the authors investigate the mechanisms\nfor enhanced female fertility in the planthopper Laodelphax striatellus induced by\nWolbachia. Two of their central conclusions are that 1. Wolbachia increases female\nfertility by increasing mitochondrial activity and that 2. that the riboflavin produced\nby Wolbachia is responsible for the increase in female fertility. While some of the\nfindings provide important insights I am not fully convinced by these main\nconclusions of the study. Conclusion 1 is based on the data from Figures 2 (showing\nthat expression of genes relating to mitochondrial function are upregulated in the\nWolbachia-infected WI line) and 3 (showing that mitochondrial function as measured\nby ATP content is improved). While the differences in gene expression are clear, I am\nnot convinced by a direct link between Wolbachia and mitochondrial activity which is\nbased on a single time point with low replication, rather than there being a clear\noverall pattern. For conclusion 2, the same issue arises and the experiments\nmanipulating riboflavin content only involve the uninfected line and not the WI line. I\ndo not have any major issues with the rest of the manuscript and the limitations raised\nhere could be addressed by completing a full set of treatments with greater replication.\nThe current level of replication for these experiments should also be justified,\nespecially when other experiments have n values far higher than 3-4. Please see more\ndetailed comments on these issues below.\nResponse: We sincerely appreciate the reviewer’s insightful feedback and have\naddressed the issues raised as detailed below. Our findings show that Wolbachia\nincreases female fertility by enhancing energy production, though the connection to\nmitochondria may not be as compelling. Consequently, we have revised the\nmanuscript to incorporate these findings and perspectives. Additionally, we have\nsupplemented data on manipulating riboflavin content in the WI line (Fig. S7). For the\nnumber of replicates, we have provided explanations below.\nLine 40 – In what way is CI harmful to the host that carries Wolbachia? CI can be\nbeneficial by giving a relative advantage to females that are infected.\nResponse: The logic is that CI is beneficial to female insects infected with Wolbachia,\nbut harmful to those not infected with the symbiont. As a result, it has an overall\ndetrimental effect on the population (Jiggins and Hurst, 2011).\nJiggins FM, Hurst GD. 2011. Microbiology. Rapid insect evolution by symbiont\ntransfer. Science, 332:185–186.\nLine 42-45 – The phrasing here is misleading because it implies that beneficial effects\n(either general or environment specific) on fecundity are a general feature of\nWolbachia strains. However this is only the case for some systems. In others such as\nmosquitoes, and especially in recent Wolbachia-host associations (such as deliberate\ntransfers through microinjection) Wolbachia can have clear costs to fecundity. The\nauthors should make it clear that effects of Wolbachia can be extremely host and\nstrain-specific and the insights of this study may not apply to other systems.\nResponse: Thanks for pointing this out. We have revised the text to be more precise.\nFigure 1. Panels a, d and e all show egg counts- are these from three separate\nexperiments done with different methods? There is no information in the methods\nabout how these comparisons differ.\nResponse: The data shown in Figures 1a, d, and e come from three independent\nexperiments, all conducted using similar methods. The primary difference lies in the\ntime intervals during which the eggs were recorded. This distinction is clarified in the\nfigure legend.\nAlso for Figure 1, it would be useful to present individual data points on a scatter dot\nplot rather than means and SEMs. Bar charts can be misleading because they obscure\nthe distribution of the data.\nResponse: We have replaced the bar charts with scatter plots to better represent the\nindividual data points, as per the reviewer’s suggestion. For data involving too many\ndata, such as in Figure 1d, we have kept the mean and standard error of the mean\n(SEM) for clarity. Additionally, we have included a detailed summary of the data,\nincluding the minimum (Min), maximum (Max), 25th percentile (Q25), 50th\npercentile (Q50), and 75th percentile (Q75), in the supplementary table\n\"Figure_1_stats.xlsx.\"\nLine 87 – There is not enough data to claim that ovaries grew faster in the WI line as\nthere is no quantification of this phenotype. Furthermore, the mature oocytes for WU\nlook larger than WI at Day 5 in the images in Figure 1. Is this because the scales are\ndifferent? The image resolution is too low for me to be able to tell.\nResponse: We quantified ovarian development by counting the number of mature\noocytes in the developing ovary. A higher count of mature oocytes indicates greater\novarian maturity. We have re-photographed the ovaries at different developmental\nstages using larger, clearer scales (Fig 1g).\nLine 151 – The content of this section does not match the claim in this subheading. In\nFigure 3 the authors show higher ATP content in WI females compared to WU\nfemales early on, then the opposite pattern at day 5. There is no clear pattern of\nimproved ATP content in WI and overall these effects are not convincing due to a low\nlevel of biological replication (typically n = 4). The effects of rotenone also do not\nappear to be substantial and only the WI line is measured. In figure 3d there is no\nquantification of oocyte developmental rates (only single representative images).\nFinally, the authors do not find any clear differences between WI and WU in terms of\nmitochondrial energy production (Figures 3f-g and supplements), with the claims in\nline 177 being based only on a single time point for complex I with n = 3. Again, in\nthese comparisons the authors should present individual data points rather than means\nand SEM only. Overall I am not convinced from the experiments here of a direct link\nbetween Wolbachia presence and mitochondrial function.\nResponse: The reviewer's comments cover a variety of aspects. I have organized the\ninformation and provided answers to the following points in the hope of resolving the\nconcerns.\nOur data indicate that WI females exhibit higher ovarian ATP levels early after\nemergence (day 3), which is necessary for supporting rapid ovarian development. By\nday 5, the ATP levels decrease, likely reflecting a more stabilized developmental state.\nThe observed reversal of ATP levels in WI females at day 5 is not inconsistent with\nthis interpretation. Additionally, the most pronounced differences in ATP content,\ncomplex I activity, and riboflavin levels between WI and WU females occur in newly\nemerged individuals (Fig. 3b, Fig. 4a).\nWe agree with the reviewer that the rotenone treatment, which inhibits complex I\nactivity, showed limited effects. To address this, we have supplemented this\nexperiment with knockdown of ATP synthase (ATPeF1B) gene expression to further\ninhibit energy production. This knockdown resulted in decreased energy production\nand delayed ovarian development, as shown in Fig. 3i-l.\nWe measured the rate of oocyte development in terms of the number of mature\noocytes as a quantitative criterion. Given the experimental focus, it might not be\nessential to extend this test to the WU group at this stage.\nWe acknowledge that the biological replication (n = 4) in some experiments, such\nas ATP content and complex I activity, is on the lower end, especially for enzyme\nactivity measurements, which are technically demanding and costly. Each\nmeasurement involves multiple individuals, with at least five individuals making up a\nsingle sample. Thus, the three to four biological replicates we used represent a\nsignificant amount of data and are sufficiently representative for these analyses.\nThe bar charts showing means and SEM have been replaced with dotted plots in\nthe revised manuscript.\nWe propose that Wolbachia enhances female fertility primarily by elevating\nenergy production (e.g., ATP content), rather than through direct modulation of\nmitochondrial function to avoid confusion. This distinction is now emphasized in the\nrevised manuscript to avoid confusion.\nLine 162 – The references to Fig 4c and 4d should be to figure 3.\nResponse: Thank you for the correction. We have revised the text.\nLine 197-199 – This claim is not supported- WU individuals performed worse than\nWI across all three diets- although the difference on the diet containing riboflavin is\nnot significant the WU line still has lower survival. To claim that\nWolbachia-synthesized riboflavin improves survival would require testing for an\ninteraction between Wolbachia status and diet. Based on the survival curves it looks\nlike WU generally just has lower fitness rather than there being a specific effect of\nriboflavin.\nResponse: Our Cox proportional hazards analysis shows that both Wolbachia status\nand diet significantly affect survival (χ² = 12.41, P = 0.0004; χ² = 18.09, P = 0.0001).\nWU individuals have lower survival across all diets, but riboflavin supplementation\nsignificantly improves their survival, bringing it closer to WI levels. There was no\nsignificant interaction between Wolbachia status and diet (χ² = 0.6084, P = 0.7377),\nbut WU planthoppers are more sensitive to riboflavin deficiency, as seen in the\nincreased survival with riboflavin supplementation. WI survival was unaffected by\nriboflavin supplementation (Likelihood ratio test = 1.4, P = 0.236). The key\nobservation is that riboflavin supplementation improves the survival and growth of\nWU individuals, indicating that these planthoppers are more dependent on riboflavin\nfor optimal fitness, in contrast to WI planthoppers, which are less sensitive to\nriboflavin status.\nLine 203 and Figure 4 – It is clear from this experiment that riboflavin\nsupplementation improves emergence/mt function/oocyte maturation in the WU line,\nbut it is unclear why only the WU line was tested. If Wolbachia-synthesised\nriboflavin is important, then you would expect to see a more pronounced improvement in the\nWU line compared to WI.\nResponse: Our initial focus was to investigate the role of riboflavin in ovarian\ndevelopment in the WU line, particularly in the absence of Wolbachia. We observed a\nmore significant improvement in the WU line following riboflavin supplementation,\nsuggesting that Wolbachia-synthesized riboflavin plays a key role in promoting\novarian development.\nThe reviewer’s comment is reasonable, and to address the concern, we extended\nthe experiment to include the WI line. Riboflavin supplementation did not promote\novarian development in WI individuals. Instead, it resulted in decreased ATP\nproduction, which contrasts with the findings in the WU line. This difference could be\ndue to excess riboflavin causing cellular damage, leading to reduced ATP production\n(Fig S7)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done a good job to revise the manuscript. They have appropriately addressed all my concerns. I recommend it to be published in this journal.", "reviewer_2": "I thank the authors for addressing my comments in detail and performing additional experiments to better support their key conclusions. I believe this has substantially improved the manuscript and I have no further suggestions."}, "author_response": "Point-by-point Response to Reviews\nReviewer #1 (Remarks to the Author):\nThe authors have done a good job to revise the manuscript. They have appropriately\naddressed all my concerns. I recommend it to be published in this journal.\nOur response: We thank you very much for your positive comments and\nrecommendation.\nReviewer #2 (Remarks to the Author):\nI thank the authors for addressing my comments in detail and performing additional\nexperiments to better support their key conclusions. I believe this has substantially\nimproved the manuscript and I have no further suggestions.\nOur response: We are very grateful for your positive comments."}]

Decision letter