Developmental plasticity enables an intestinal tapeworm to adapt to dietary stress

Domain: chemistry
Published: 2026-02-20
Editorial outcome: accept

Abstract
Diet is one of the strongest ecological forces shaping the gut environment, yet its impact on intestinal worms (helminths) remains poorly understood. The helminth Hymenolepis diminuta is a suitable model for investigating how lifestyle changes in modern societies may disrupt host–helminth relationships. Here we show that dietary fiber availability shapes the developmental trajectory and life strategies of H. diminuta in a stage-dependent manner. Fiber deprivation at the time of host colonization leads to developmental arrest, manifested by reduced growth, absence of reproduction, and transcriptional changes consistent with suppressed development. This state is accompanied by diet-dependent remodeling of the host small intestinal microbiota and metabolome: whereas fiber-rich diets support fermentative microbial communities and a chemically diverse intestinal environment, the Western diet promotes dysbiotic profiles with reduced fermentation capacity and a more pro-inflammatory immune response. In contrast, adult H. diminuta that reach maturity in hosts maintained on a fiber-rich diet exhibit a reversible, estivation-like suppression of reproduction during short-term fiber deprivation, with full restoration of egg production following dietary recovery. Together, these findings indicate that dietary transitions associated with industrialized lifestyles can redirect helminth developmental programs and host–helminth–microbiome interactions, with implications for helminth persistence and potential therapeutic applications.

Introduction
The gut is a complex ecosystem where the host coexists with a wide array of microbial and macrobiotic symbionts1. While research has long focused on bacteria, the gut community also harbors viruses, archaea, fungi, protists, and, in some cases, multicellular eukaryotes such as helminths2–5. These often-overlooked members are now recognized as integral to intestinal ecology, able to reshape microbial balance and modulate host immunity through distinct metabolic and immunological pathways2,6.

Intestinal helminths are increasingly recognized as macrosymbionts with strong immunomodulatory capacity. Once a routine part of the human gut ecosystem, they have largely vanished from Western populations7. Their presence can reshape both immunity and bacterial communities, often enriching beneficial Clostridia groups while suppressing pro-inflammatory taxa such as Bacteroides and Proteobacteria2. Helminths, microbiota, and the host immune system operate as an interdependent network, where the impact of each partner depends on the others8. Crucially, specific bacterial taxa can amplify or dampen helminth-driven immune regulation, revealing the bidirectional and context-dependent nature of these interactions9,10.

The decline in helminths is widely attributed to improved sanitation, urbanization, and mass anthelmintic use5,11,12. However, shifts in dietary patterns may also play a decisive role by altering the ecological conditions required for their persistence. Western diets, typically low in microbe-accessible fiber, drive profound changes in gut fermentation profiles and substrate availability, potentially impairing the survival of eukaryotic symbionts13,14. While dietary effects on bacterial communities are well documented, the consequences of host diet for non-bacterial gut residents—particularly helminths—have been explored only in a limited and system-specific way. Emerging studies indicate that diet can influence helminth persistence and developmental outcomes, including tendencies toward hypobiosis15–17, yet this work remains in its early stages and confined to specific experimental systems. Thus, the effects of complex diets on helminth development in vivo are still only partly understood.

In addition, modern diets alter key physicochemical parameters of the gut—such as luminal pH, oxygen (redox) gradients, mucus architecture and transit time—that define ecological niches. These features shape microbial composition and activity across intestinal regions18,19. Experimental work further shows that dietary context profoundly reconfigures community assembly, keystone functions and metabolic outputs, with strain–strain relationships and pH modifications varying across carbohydrate environments19. Such nutritional selection pressures likely drive microbial evolutionary shifts under Westernized conditions14 and may contribute, together with other aspects of modernization, to the documented decline of some intestinal eukaryotic symbionts, including helminths, in industrialized populations3,7.

This renewed ecological perspective has also revealed how helminths, through reshaping host-associated microbial networks, modulate immune and metabolic outcomes in ways that can protect against diet-induced pathology20. The rat tapeworm Hymenolepis diminuta is among the most promising model helminths, combining potent immunomodulatory and anti-inflammatory effects with low pathogenicity, a well-defined life cycle, and ease of laboratory maintenance5,21. Preclinical studies show protective effects against chemically induced inflammation, allergies, neuroinflammation, and behavioral disorders21–24. In rats, colonization alleviates colitis, reduces expression of Il1b and Tnf, increases Il10, and promotes mucosal repair23, while shifting the microbiota toward an anti-inflammatory profile, yet these changes are reversed upon decolonization9,25–27. Case reports of self-treatment for conditions such as Inflammatory Bowel Disease, migraines, and ADHD report good safety and tolerance21,28,29. Yet, therapeutic efficacy is inconsistent, shaped by microbiota composition, inflammatory state, and dietary context. Antibiotic treatment abolishes these benefits9, underscoring the role of host–microbiota–helminth interactions8. Diet emerges as a key modulator, influencing both gut ecology and helminth development. From an evolutionary perspective, fiber-depleted industrial diets may have imposed selective pressures contributing to the disappearance of eukaryotic gut symbionts13,21. Early experimental work showed that H. diminuta physiology can respond to nutritional conditions, but these studies relied mainly on isolated nutrients and simplified settings30. Here, we show that dietary composition is a critical determinant of the development, physiology, and persistence of the intestinal tapeworm H. diminuta. By comparing a Western-typed diet rich in fat and refined sugar but lacking microbe-accessible fiber with a complex, fiber-rich diet, we demonstrate that fiber deprivation profoundly constrains tapeworm growth, reproductive maturation, and transcriptional activity. These diet-dependent effects coincide with marked restructuring of the host gut ecosystem, indicating that modern low-fiber dietary patterns destabilize long-standing host–helminth ecological relationships. Together, our findings identify dietary fiber as a key ecological factor governing the viability and functional integration of gut-resident eukaryotic symbionts.

Results
Diet-driven plasticity in tapeworm development
Experiment 1 (Fig. 1A), which tested the effect of host diet on the development of Hymenolepis diminuta, revealed marked diet-dependent differences in helminth morphology and reproductive output. Rats were inoculated after diet change on day 30 (Fig. 1A). Tapeworms from rats maintained on the Accessible Fiber diet exhibited a normal colonization rate (100%; n=16) and fully mature morphology, characterized by normal length (i.e., 20–60 cm in rats), fully developed copulatory organs, and egg-filled uteri in the posterior proglottids (Fig. 2A, C, E). In contrast, H. diminuta recovered from rats on the Western (fiber-free) diet showed a reduced colonization rate (50%; 16 out of a total of 32 rats) and arrested development, displaying an immature phenotype (Fig. 2B, D). Among colonized hosts, tapeworms from Western-diet rats were dramatically shorter and undeveloped than those from Accessible Fiber fed rats (Western diet: 2.69±1.41 cm, n=16; Accessible Fiber diet: 20.81±2.74 cm, n=16; mean difference=18.13±0.77 cm 95% CI [16.53, 19.72]; Welch’s t-test, t(22)=23.49, p<0.0001, η²=0.96) (Fig. 2F; Source data are available on Figshare https://doi.org/10.6084/m9.figshare.26038633).

Using Sheather’s flotation, H. diminuta eggs were detected in the feces of rats maintained on the Accessible Fiber diet, whereas no eggs were found in those on the Western diet during the patent period (i.e., ≥20 days post-colonization, when adult tapeworms are typically fully developed). Unexpectedly, necropsy on day 60 (i.e., 30 days post-inoculation) consistently revealed the presence of immature tapeworms in Western-diet rats. Further morphological evaluation confirmed arrested development, with posterior proglottids containing only immature reproductive organs and lacking a developed uterus (Fig. 2B, D).

For clarity, colonization is defined here as the presence of H. diminuta in the host gut, irrespective of reproductive activity. In rats fed the Accessible Fiber diet, colonization was confirmed by egg shedding, whereas in rats on the Western diet, no eggs were detected, but endpoint necropsies consistently revealed stunted immature tapeworms. Thus, the definition of colonization was consistent across groups, while the method of confirmation differed depending on diet.

Experiment 2 (Fig. 1B) tested whether the reproductive effects of a temporary Western diet exposure are reversible in adult tapeworms. A 90-day experiment with three dietary regimens was conducted, with all rats initially colonized on the Accessible Fiber diet (n=6 per group; total n=18); the diet shift was introduced when patency was confirmed (Fig. 1B). Egg output was monitored throughout and revealed that reproductive activity can indeed be restored by dietary manipulation. In the experimental recovery group, colonized rats were first maintained on the Accessible Fiber diet for 50 days (30 days of exposure plus a 20-day prepatent period), then switched to the Western, fiber-free diet for 10 days, and finally returned to the original fiber-rich diet for 30 days (Fig. 3).

Rats kept continuously on the Accessible Fiber diet maintained high and stable egg counts during the patent period (egg counts between days 21 and 50: 999.7±82.95 eggs/g feces). Switching to the Western diet on day 50 triggered a rapid decline, culminating in complete cessation of egg shedding by day 55. Reintroduction of the Accessible Fiber diet restored egg production on between days 73-75 (egg counts from day 73—251.8±71.34 eggs/g during recovery), with all returning rats resuming egg shedding at levels approaching those of continuous control group (egg counts from day 73—737.8±55.54 eggs/g), indicating recovery of reproductive capacity (Fig. 3). In contrast, rats maintained on the Western diet from day 50 until the end of experiment exhibited egg count 0.

Repeated-measures negative binomial generalized linear modeling revealed a strong group×time interaction (χ²(2)=290.83, FDR-adjusted p<0.001) in all groups (n=6 per group). The Accessible Fiber group showed stable egg output over time (slope=−0.00332 eggs/day, 95% CI [−0.01299, 0.00635]), while both groups exposed to the Western diet exhibited steep declines (Western diet: slope=−0.20480, 95% CI [−0.22832, −0.18128]; recovery group during Western diet: slope=−0.03617, 95% CI [−0.04434, −0.02801]). Following fiber reintroduction, egg production resumed in this group as indicated by a significantly attenuated temporal decline relative to the Western diet group (slope=0.16862, 95% CI [0.14379, 0.19346], Z=13.31, p<1×10−10; Fig. 3).

Transient fluctuations in egg counts during days 25–35 resulted in some early statistical differences; however, these occurred while all rats were maintained on the same diet and were therefore not considered biologically meaningful. Following dietary transitions, significant differences between the continuous Accessible Fiber group and both the Western and recovery groups on days 54–57 (model-based contrasts: Z=3.1–6.1, FDR-adjusted p<0.001). During the recovery phase, egg counts in the recovery group remained significantly lower than those of the continuous Accessible Fiber group on days 73 and 75 (Z=4.2–4.7, FDR-adjusted p<0.001), reflecting the delayed resumption of egg production (Fig. 3).

Within-group analyses using the same modeling framework supported these findings: egg output remained stable in the continuous Accessible Fiber group (all comparisons p>0.35), declined rapidly and did not recover within the experimental timeframe under the Western diet (p<0.001), and was restored after fiber reintroduction (recovery group: significant reduction during Western diet, p<0.001; recovery to pre-transition levels, p=0.50; Fig. 3; Source data are available on Figshare https://doi.org/10.6084/m9.figshare.26038633).

Transcriptomic adaptations of tapeworm to dietary stress
Comparative transcriptomic profiling of H. diminuta, performed on three biological replicates per dietary group, identified pronounced diet-associated differences in gene expression between tapeworms recovered from rats fed the Western (fiber-free) diet and those fed the Accessible Fiber diet. Using a conservative threshold (FDR<0.01, |log₂FC|>1), we detected 691 upregulated and 786 downregulated genes in tapeworms from the Western diet group relative to the Accessible Fiber diet group (Fig. 4, Supplementary Figs. 1–3).

Specifically, genes showing reduced expression under the Western diet were enriched for functional categories associated with multicellular organism development, including components of the WNT signaling pathway (9 genes; Supplementary Fig. 4), as well as genes involved in mitosis and meiosis (zyg-11, 25 centrosome components and 3 recombination nodule components) (Supplementary Data 1). In addition, reduced expression was also observed for genes annotated to muscle function (e.g., myosin metabolism; 4 genes) and energy metabolism pathways (e.g., glucose metabolism; 2 genes). Conversely, genes with relatively higher expression in tapeworms from the Western diet were enriched for purine and pyrimidine metabolism (11 genes), transmembrane transporter activity (17 genes), and cell–cell junctions (16 genes) (Supplementary Fig. 1).

Genes related to cellular stress responses were also differentially expressed. Thirteen genes were annotated as oxidative stress regulation, detoxification, and mitochondrial signaling. These include genes related to the modulation of reactive oxygen species (ROS), such as voltage-gated proton channel genes (two upregulated, three downregulated) and cytochrome b5 reductase genes (two upregulated). Detoxification-associated genes with glutathione transferase activity were downregulated (five genes), and the tryptophan-rich sensory protein (tsp) gene, a homolog of the mitochondrial benzodiazepine receptor, was also downregulated (Supplementary Data 1).

Small intestinal microbiota shaped by diet and tapeworm
Microbiome analyses showed that diet dominated small intestinal communities, whereas effects of H. diminuta were subtle and diet dependent. The total number of reads after quality filtering was 5,407,746 (min=74,053; max=153,034; median=111,752), which clustered into 4180 ASVs. Rats fed the Accessible Fiber diet showed significant enrichment of bacterial taxa typically associated with gut homeostasis and beneficial metabolic activity, such as fiber fermentation. These included members of the order Lactobacillales, such as Lactobacillus, Ligilactobacillus, and Limosilactobacillus, as well as Turicibacter and taxa within the order Erysipelotrichales (Fig. 5A). In contrast, the Western diet promoted the expansion of multiple taxa linked to dysbiosis and host stress, including genera such as Streptococcus, Staphylococcus, Clostridium sensu stricto 1, and Romboutsia, affiliated with the orders Clostridiales, Staphylococcales, Burkholderiales, and Peptostreptococcales–Tissierellales (Fig. 5A).

LEfSe analysis further revealed that shifts associated with H. diminuta colonization were strongly diet-dependent, with no enriched

Methods

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The stated goal of this study is to examine the role of diet in inducing aestivation in the rat tapeworm, Hymenolepis diminuta.\nTo do this, the authors performed a series of experiments testing the effects of host diet [Western (W) vs. Accessible fiber (A)\ndiets] on parasite morphology and gene expression as well as the host microbiome, metabolome and immunity. Questions\nabout how host diet might influence interactions between hosts and their parasites are of considerable interest and it is clear\nfrom this study that there are complex and intriguing interactions occurring among host, parasite and microbiome that are\nmediated by diet. However, the experimental design and analyses presented in this study do not adequately disentangle\nthese complexities. Importantly, the study design and results do not appear to support the conclusions that are being drawn,\nand the findings are not adequately contextualized within the existing literature. More detailed comments are provided\nbelow.\nMajor comments\nIntroduction\nThe introduction requires deeper context to introduce the motivating questions and their relevance/significance within the\nexisting literature. For example, it seems relevant to discuss the fact that H. diminuta can infect people as part of the\nmotivation for looking at the effects of a Western diet. A brief discussion of the biology of this parasite in humans seems like\nnecessary context for this study.\nEven more important, the paper would benefit from a discussion of what is already known about the interplay between host\ndiet, microbiome, metabolome, immunity, and helminth parasitism. This includes information from past studies on H.\ndiminuta, which are alluded to on line 81 but never described, as well as from other systems, including other host-helminth\nsystems (e.g. domestic livestock systems).\nFinally, the phenomenon of aestivation needs to be better defined and characterized given similar processes known to occur\nin helminths. Helminths show a range of responses to stress that are similar to aestivation (e.g., arrested/delayed\ndevelopment, hypobiosis, dauer) and some of these processes have been previously linked to host diet. For example,\nparasitic nematodes can delay their growth or reproduction in response to host diet (e.g., Carvalho et al 2023\nhttps://doi.org/10.3390/ruminants3040033). If aestivation is a distinct biological phenomenon from these other well-described\nhelminth stress responses, this should be clearly explained, and the introduction and/or methods should describe how\naestivation is being qualitatively or quantitatively identified in this paper.\nMethods\nIt is difficult to understand the experimental design and data analysis without reading the supplementary materials.\nImportantly, key details on study design and statistical analyses need to be presented in the main text for readers to\nunderstand what was done and why. For example, in lines 100-106, the sample size of rats per treatment, dosage of\nhelminths administered, and approach to retrieve and quantify adult worms should be clearly described, along with the\nnumber of adult worms retrieved at the endpoint of the experiment for each group. Including the figure from Supplementary\nData 2 in the main text would be helpful for visualizing Experiment 2. It is crucial that sample sizes are clearly described in\nthe main text (in the methods/alongside the results) so the power of the approach can be evaluated.\nCurrently, Experiment 2 is presented as a test of the reversibility of the “aestivation” phenomenon observed in Experiment 1\n(lines 106-108), however, it is not clear that Experiments 1 and 2 are testing the same phenomenon. In Experiment 1, H.\ndiminuta cysticeroids were administered to rats while on the Western diet or Accessible Fiber diet, and the Western diet\nappeared to affect their development into adult worms. In Experiment 2, H. diminuta cysticeroids were administered to rats\nwhile on the Accessible Fiber diet, allowed to develop into adult worms, and then rats were switched to the\nWestern diet. Thus, Experiment 1 appears to test the effects of poor host nutrition on worm maturation from cysticeroids into adults, while\nExperiment 2 tests how poor host nutrition affects adult worms that have already matured on an accessible fiber host diet. To\ntest the stated question about reversibility of the developmental effects observed in Experiment 1, cysticeroids would need to\nbe administered while rats are fed on a Western diet, then switched to the Accessible Fiber diet. This mismatch between the\nexperiment that was actually done and the claims made needs to be addressed. At present, it does not appear that the\nauthors have directly tested the reversibility of aestivation.\nAn explanation should be given for why particular diets were chosen and some discussion is needed of the key differences\nin diet components that were the target of the study and why. A list of ingredients as outlined (lines 92-96) is insufficient.\nResults & discussion\nThe combination of results and discussion into a single section made the study findings and conclusions somewhat hard to\nfollow. One recommendation is to disaggregate these sections. Furthermore, statistics (name of statistical test, sample sizes,\ntest statistics, p-values) should be reported in the main text as support (e.g., Lines 112-116, 163-192, 207-216, 224, 235,\n237). Currently, it is difficult to link the statistical methods described in the supplementary material to the results outlined in\nthe main text and there are cases where the statistical methods described in the supplement appear not to entirely explain\nthe described results. There are also cases where incorrect statistical approaches seem to have been used (e.g., Figure 2,\nFigure 5). It should be clear which statistical methods were used for every result presented and claims should not be made\nwithout statistical evidence (e.g. lines 112-117, 147-150).\nHow was worm colonization determined for the different groups being compared and what does the term “colonized” mean\n(line 118)? Is a colonized rat an animal that was initially inoculated with worms, that shed eggs, or that had adult worms at\nthe endpoint of the experiment? Depending on the definition of colonization, interpretation of these results may change.\nIn general, many results have been overinterpreted or inaccurately represented. For example:\nLines 112-117: If the sample size for worm morphology analyses is as low as 2 worms total from each of the 2 diet\ntreatments, as stated in Supplementary Methods (Line 97), then the morphological results described here are not\nmeaningful.\nLines 147-150: Without reporting adult worm counts or mortality rates, it is not possible to draw conclusions about differential\nworm mortality. Additionally, without mortality data, it is difficult to determine whether the phenomena observed here are due\nto developmental changes or differences in mortality or establishment between diet treatments.\nLines 211-213: These results do not seem to match the figure caption or reported statistics, which suggest that the made\ncomparison is between diets, not within them. Reporting statistics in the main text would aid in clarity and support.\nLines 250-253: Host immune responses seem like a critical part of the study but are only described briefly. Including a figure\nwith immune data in the full text seems necessary to interpret these statements. All patterns observed in both the Western\ndiet and Accessible Fiber diets should be described. For example, on a Western diet, colonization also had effects on IL4\nand IFN-g (Supplementary Data 10) that are not referred to at all in the text. Full, transparent reporting of these results is\nnecessary for interpretation.\nFigure 2: This figure truncates some of the time series data presented in Supplementary Figure 3. Presenting the full time\nseries data on egg shedding shown in the supplementary figure and explaining the observed variation between groups\nwhile fed the same diet (i.e., days 21-49) would be more transparent. Some of these differences appear significant and are\nnot currently explained in the text. Additionally, the methods or results should explain and show the specific statistical tests\nused to obtain the significance differences shown on the graph, and explain which specific timepoints, treatments, or\nindividuals are being compared. Currently, the meaning of these significance levels is unclear.\nFigure 4: There appears to be inaccuracy in the reporting or visualization of results in this figure. Comparisons in 4A and 4B\nare unclear and should be indicated on the figure. Visually, figure 4B does not seem to show a statistically different effect of\ncolonization in the A-diet versus the W-diet (the bars on the graph appear identical), although the figure caption reports one.\nFigures 4C and 4D do not match the findings reported in lines 211-216. These figures also show that each diet is more\nsimilar to itself, but do not make within-diet comparisons.\nFigure 5: The results reported in the text (Lines 217-221) contradict the statistics presented in this figure caption. While the\neffect sizes vary, colonization has statistically significant effects on the metabolome for both diets, not just the Western diet.\nAdditionally, to test whether H. diminuta alters the small intestinal bacteriome in a diet-dependent manner (lines 530-531)\nrequires testing for an interaction between diet and colonization on these indices, which does not appear to have been done.\nMinor comments\nLines 77-80: The relevance of therapeutics for H. diminuta is unclear. Negative impacts on humans or animals have not\nbeen described.\nLine 114: The statement that worms on the Western diet have a “typical morphology” and “normal length” requires a clear,\nquantitative description of typical morphology.\nLines 135-139: It’s essential to know the average total worm burden in each group at the end point of Experiment 1 in order\nto interpret this statement. Additionally, it is unclear whether these lines refer to results from Experiment 1 or Experiment 2.\nLines 144-145: The conclusion that “tapeworms markedly reduce their metabolic requirements” in response to a Western\ndiet requires evidence not currently presented in the text.\nLines 288-299: This is the first time that H. diminuta is described as a human pathogen. This comes too late in the\nmanuscript to be a convincing broader extension of the work.\nFigure 3: This figure is difficult to interpret in its current form; it should highlight specific patterns or genes of interest for\ngreater clarity.\nLine 243-247: Fructose is the key factor driving separation between diet groups (according to line 226). Yet in line 245,\nchanges in the metabolome are discussed in the context of glucose, sucrose, and maltose. Providing some interpretation of\nthe key fructose result would be helpful.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "The authors present a well-designed study investigating how two distinct diets, one fiber-rich and the other representative of\na Western dietary pattern, affect the development of the intestinal tapeworm Hymenolepis diminuta in a rat model. Their data\nclearly show that the Western-like diet slows down development of this organism in a reversible manner. They hypothesize\nthat the parasite gets into an aestivation state due to factors related to the Western diet and the influence of this diet on the\nmicrobiota. They support their hypothesis with gene expression and metabolomics data. These data are insightful. The main\nfinding is compelling and counterintuitive: not only does it suggest a hidden link between Western lifestyle and reduced\ntapeworm infections, but it also raises the possibility that tapeworms may evade detection in modern societies due to their\nunderdeveloped forms.\nThe limitation of the study is that, beyond Figure 2, the analyses become relatively superficial. The causal connections\nbetween tapeworm physiology and dietary components (or diet-derived metabolites) are not definitively established and\nappear to fall outside the current scope of the paper. Furthermore, the study does not address how variations in diet\ncomposition might alter the outcome. For instance, how would a modified Western diet formulated to reduce fructose levels\nin the gut impact the observed effects? Fructose emerges as a potentially important metabolite in the analysis, yet its role\nremains speculative without experimental testing. Whether the study meets the threshold for publication in this journal\nwithout such deeper mechanistic or dietary exploration is ultimately an editorial decision.\nMinor comments:\nPage 3, lines 93-97: Please clarify what the reported percentages refer to (mass percentage, caloric contribution, etc.)\nPage 7, lines 213-216: The conclusion that dietary fiber absence leads to \"destabilization\" of the microbiota is not fully\nsupported by Figure 4. Please elaborate on how this conclusion is derived from the data.\nPage 7, lines 237-239: The claim that specific diacylglycerols and amino acids are present only in animals with H. diminuta\nis not directly supported by the cited figures. Please provide metabolite-level information or clarify the connection."}, "author_response": "RESPONSES TO REVIEWERS` COMMENTS\nREVIEWERS #1 & #2:\nR1 & 2 comment: The stated goal of this study is to examine the role of diet in inducing aestivation in the\nrat tapeworm, Hymenolepis diminuta. To do this, the authors performed a series of experiments testing the\neffects of host diet [Western (W) vs. Accessible fiber (A) diets] on parasite morphology and gene expression\nas well as the host microbiome, metabolome and immunity. Questions about how host diet might influence\ninteractions between hosts and their parasites are of considerable interest and it is clear from this study\nthat there are complex and intriguing interactions occurring among host, parasite and microbiome that are\nmediated by diet. However, the experimental design and analyses presented in this study do not\nadequately disentangle these complexities. Importantly, the study design and results do not appear to\nsupport the conclusions that are being drawn, and the findings are not adequately contextualized within\nthe existing literature. More detailed comments are provided below.\nA: We thank the reviewers for their constructive and thoughtful comments. We believe that some of the\ninitial concerns may have arisen from the fact that our manuscript was directly transferred from the original\nsubmission to Nature, where strict formatting limitations required us to condense the main text and move\nseveral key sections to the Supplementary Materials. In the revised version, we have reintegrated these\nimportant details into the main text and substantially expanded the contextualization of our findings within\nthe existing literature. We have also clarified how the experimental design and analyses support the\nconclusions, thereby addressing the reviewers’ concerns regarding the interpretation of host–helminth–\nmicrobiome interactions.\nR1 & 2 comment: Introduction\nAuthors` note – see response for this section below.\nThe introduction requires deeper context to introduce the motivating questions and their\nrelevance/significance within the existing literature. For example, it seems relevant to discuss the fact that\nH. diminuta can infect people as part of the motivation for looking at the effects of a Western diet. A brief\ndiscussion of the biology of this parasite in humans seems like necessary context for this study.\nEven more important, the paper would benefit from a discussion of what is already known about the\ninterplay between host diet, microbiome, metabolome, immunity, and helminth parasitism. This includes\ninformation from past studies on H. diminuta, which are alluded to on line 81 but never described, as well\nas from other systems, including other host-helminth systems (e.g. domestic livestock systems).\nFinally, the phenomenon of aestivation needs to be better defined and characterized given similar\nprocesses known to occur in helminths. Helminths show a range of responses to stress that are similar to\naestivation (e.g., arrested/delayed development, hypobiosis, dauer) and some of these processes have\nbeen previously linked to host diet. For example, parasitic nematodes can delay their growth or\n1\nreproduction in response to host diet (e.g., Carvalho et al 2023\nhttps://doi.org/10.3390/ruminants3040033). If aestivation is a distinct biological phenomenon from these\nother well-described helminth stress responses, this should be clearly explained, and the introduction\nand/or methods should describe how aestivation is being qualitatively or quantitatively identified in this\npaper.\nA: We thank the reviewers for this valuable suggestion. The introduction has been fully revised to provide a\ncomprehensive overview of the biology of Hymenolepis diminuta, its current relevance in the context of\nhelminth therapy, and the known interactions among host diet, microbiome, metabolome, immunity, and\nhelminth infections (lines 60–114). In addition, references describing studies on H. diminuta as well as\nrelevant findings from other host–helminth systems have been incorporated.\nThe terminology and conceptual distinctions between aestivation and other stress responses in\nhelminths, such as hypobiosis, dauer, arrested development etc are now addressed in the opening part of\nthe Discussion, where they are more logically placed and compared directly with the relevant literature on\nmodel helminths (lines 275–351).\nR1 & 2 comment: Methods\nIt is difficult to understand the experimental design and data analysis without reading the supplementary\nmaterials. Importantly, key details on study design and statistical analyses need to be presented in the main\ntext for readers to understand what was done and why. For example, in lines 100-106, the sample size of\nrats per treatment, dosage of helminths administered, and approach to retrieve and quantify adult worms\nshould be clearly described, along with the number of adult worms retrieved at the endpoint of the\nexperiment for each group. Including the figure from Supplementary Data 2 in the main text would be\nhelpful for visualizing Experiment 2. It is crucial that sample sizes are clearly described in the main text (in\nthe methods/alongside the results) so the power of the approach can be evaluated.\nA: We fully agree with this comment. This discrepancy arose because the manuscript was originally\nprepared for submission to Nature, where formatting constraints led to a condensed Methods section and\nreliance on Supplementary Materials. Here, in the revised version, the text has been thoroughly adapted to\nmeet the guidelines of Nature Communications. All requested information, including sample sizes, helminth\ndosage, and methods for worm retrieval and quantification, has been incorporated into the main text (see\nlines 459-523; or full M&M section 459-681), and the designs of both experiments are now clearly\nvisualized in the revised Figure 1.\nR1 & 2 comment: Currently, Experiment 2 is presented as a test of the reversibility of the “aestivation”\nphenomenon observed in Experiment 1 (lines 106-108), however, it is not clear that Experiments 1 and 2\nare testing the same phenomenon. In Experiment 1, H. diminuta cysticeroids were administered to rats\nwhile on the Western diet or Accessible Fiber diet, and the Western diet appeared to affect their\n2\ndevelopment into adult worms. In Experiment 2, H. diminuta cysticeroids were administered to rats\nwhile on the Accessible Fiber diet, allowed to develop into adult worms, and then rats were switched to the\nWestern diet. Thus, Experiment 1 appears to test the effects of poor host nutrition on worm maturation\nfrom cysticeroids into adults, while Experiment 2 tests how poor host nutrition affects adult worms that\nhave already matured on an accessible fiber host diet. To test the stated question about reversibility of the\ndevelopmental effects observed in Experiment 1, cysticeroids would need to be administered while rats are\nfed on a Western diet, then switched to the Accessible Fiber diet. This mismatch between the experiment\nthat was actually done and the claims made needs to be addressed. At present, it does not appear that the\nauthors have directly tested the reversibility of aestivation.\nA: We sincerely thank the reviewers for this insightful and constructive comment, which we consider key\nfor refining and strengthening the manuscript. We fully agree with the concern and, during the revision,\ncarefully considered two possible ways to address it:\nOption 1 (implemented in this revision): In the revised manuscript, we have substantially strengthened the\nconceptual and textual integration of both experiments. We placed particular emphasis on clarifying that\nthey describe two distinct yet related phenomena occurring in H. diminuta under Western diet conditions,\ndepending on the developmental stage of the helminth at which dietary stress occurs (see lines in sections\nResults – 119-1171 and Discussion – 275-310). With this revision, we have eliminated the previous\nambiguity, and the manuscript now clearly explains that Experiment 1 and Experiment 2 were designed to\ntest two different processes, directly addressing the reviewer’s concern about a mismatch between the\nexperimental design and the conclusions. We believe this change has considerably improved the clarity and\nlogic of the manuscript. At the same time, it opens an underexplored area in helminths with therapeutic\npotential, helping to explain discrepancies in clinical outcomes of helminth therapy and highlighting the\nimportance of host diet in such applications. We are confident that this solution best addresses the\nreviewers’ concerns and provides the manuscript with greater clarity and broader relevance.\nOption 2 (future direction): Alternatively, one could design a new experiment specifically to link the two\ncurrent experiments, particularly to directly test the reversibility of “aestivation” in juvenile tapeworms\ncolonizing rats under Western diet conditions. While such an experiment would be theoretically valuable, in\npractice it would represent a highly complex, long-term, and high-risk project. The experiment itself would\nrequire at least 2.5 months, preparation another 1.5 months, and several further months for sample\nprocessing and data analysis. Moreover, because the colonization success rate of rats on the Western diet\nis relatively low (~50%), the experiment would likely need to be repeated, extending the overall timeline to\nmore than one year. For transparency, we provide the outline of such a potential design below this\nparagraph (Design 1), which illustrates both the conceptual value and the practical limitations of this\napproach. Given these constraints, we consider this option beyond the feasible scope of the present study\nand instead regard it as an important avenue for future, dedicated research aimed at a deeper\nunderstanding of H. diminuta physiology under different dietary regimes and its immunomodulatory effects\non the host.\nHymenolepis diminuta aestivation under Western diet conditions. The design combines elements of\nExperiments 1 and 2, including colonization of rats with cysticercoids under Western diet, dietary\ntransitions to Accessible Fiber diet, and parallel monitoring of worm development, morphology, and egg\nproduction. This conceptual scheme is provided for transparency to illustrate the value of such an approach\nas well as its practical limitations.\nR1 & 2 comment: An explanation should be given for why particular diets were chosen and some discussion\nis needed of the key differences in diet components that were the target of the study and why. A list of\ningredients as outlined (lines 92-96) is insufficient.\nA: We agree that the rationale for diet selection should be described in greater detail in the manuscript.\nOur aim was to create a strong nutritional contrast between two extremes, a highly processed Western diet\nand a natural, hunter gatherer-like diet. This allowed us to capture clear differences in host and helminth\nresponses in a model that dramatically reflects widespread cultural changes. As the reviewer points out,\nthis approach does not allow us to determine the effects of specific components in the diet. However, the\nresults based on this approach are clinically relevant.\nIn the revised version, we have therefore expanded the explanation - aspects of the modern\nWestern diet are now described in the Introduction (lines 81-89), and the rationale for diet selection is\ndiscussed in more detail both at the end of the Introduction (lines 106–114) and at the beginning of the\nMethods section (lines 460-464). In addition, the table summarizing the composition of both diets has been\nmoved from the Supplementary Materials into the main text (Table 1) and further elaborated to highlight\nthe key components underlying the dietary contrast examined in this study.\nR1 & 2 comment: Results & discussion\nThe combination of results and discussion into a single section made the study findings and conclusions\nsomewhat hard to follow. One recommendation is to disaggregate these sections.\nA: We fully agree with this comment. The combined Results and Discussion section was a relic of the\noriginal Nature submission format. In the revised manuscript, these sections have been separated and\n4\nthoroughly revised in accordance with Nature Communications guidelines, which we believe has\nconsiderably improved the clarity and readability of the study.\nR1 & 2 comment: Furthermore, statistics (name of statistical test, sample sizes, test statistics, p-values)\nshould be reported in the main text as support (e.g., Lines 112-116, 163-192, 207-216, 224, 235, 237).\nCurrently, it is difficult to link the statistical methods described in the supplementary material to the results\noutlined in the main text and there are cases where the statistical methods described in the supplement\nappear not to entirely explain the described results. There are also cases where incorrect statistical\napproaches seem to have been used (e.g., Figure 2, Figure 5). It should be clear which statistical methods\nwere used for every result presented and claims should not be made without statistical evidence (e.g. lines\n112-117, 147-150).\nA: We fully agree with the reviewers on this important point. In the revised manuscript, the Results section\n(lines 118-271) has been thoroughly revised and all statistical details are now reported directly in the main\ntext, including the names of the tests used, sample sizes, test statistics, and p-values. This ensures that each\nresult is clearly supported by the corresponding statistical evidence.\nIn addition, the statistical analysis for egg counts (previously Figure 2, now Figure 3) has been\ncarefully re-evaluated and revised. The updated methodology is described in the Methods section (lines\n502-523), the revised results are presented in the Results section (lines 144-171), and the corrected data\nare shown in Figure 3.\nR1 & 2 comment: How was worm colonization determined for the different groups being compared and\nwhat does the term “colonized” mean (line 118)? Is a colonized rat an animal that was initially inoculated\nwith worms, that shed eggs, or that had adult worms at the endpoint of the experiment? Depending on the\ndefinition of colonization, interpretation of these results may change.\nA: We thank the reviewer for this important comment, which indeed requires clearer explanation in the\nmain text.\nIn this study, we define “colonization” as the presence of H. diminuta in the host gastrointestinal tract in\neither adult or developmentally arrested form, irrespective of reproductive activity. The method of\nconfirming colonization, however, differed between diet groups due to diet-dependent differences in\nhelminth development. In rats maintained on the Accessible Fiber diet, colonization was confirmed by\npositive egg detection using Sheather’s flotation method after the prepatent period. In rats maintained on\nthe Western diet, no eggs were detected after the prepatent period, but endpoint necropsies consistently\nrevealed stunted worms, confirming helminth presence despite the absence of reproductive maturity.\nThus, the definition of colonization was consistent across groups, while the method of confirmation\ndiffered depending on diet. This clarification has now been included in the revised Results section (lines\n137–142).\nexample:\nLines 112-117: If the sample size for worm morphology analyses is as low as 2 worms total from each of the\n2 diet treatments, as stated in Supplementary Methods (Line 97), then the morphological results described\nhere are not meaningful.\nA: We thank the reviewer for this very helpful comment as usual. The statement in the M&M section was\nan unfortunate misrepresentation. In fact, the total number of adult worms analyzed was 16 from the\nAccessible Fiber diet group (2 worms per rat from 8 rats with 100% colonization success) and 8 from the\nWestern diet group (up to 2 worms per colonized rat, with approximately 50% colonization success). This\nhas now been corrected and clarified in the Methods section (lines 559–562).\nR1 & 2 comment: Lines 147-150: Without reporting adult worm counts or mortality rates, it is not possible\nto draw conclusions about differential worm mortality. Additionally, without mortality data, it is difficult to\ndetermine whether the phenomena observed here are due to developmental changes or differences in\nmortality or establishment between diet treatments.\nA: We fully agree with the concern. In the revised manuscript, statements referring to worm mortality have\nbeen removed and the relevant sections have been carefully rephrased to avoid overinterpretation (see\nlines 120–142). The text now clearly distinguishes between reduced colonization success and\ndevelopmental arrest under the Western diet.\nR1 & 2 comment: Lines 250-253: Host immune responses seem like a critical part of the study but are only\ndescribed briefly. Including a figure with immune data in the full text seems necessary to interpret these\nstatements. All patterns observed in both the Western diet and Accessible Fiber diets should be described.\nFor example, on a Western diet, colonization also had effects on IL4 and IFN-g (Supplementary Data 10)\nthat are not referred to at all in the text. Full, transparent reporting of these results is necessary for\ninterpretation.\nA: In the revised manuscript, the immunological data have been incorporated directly into the main text,\ntogether with a dedicated figure (Figure 9). All relevant results are now systematically reported in the\nResults section (lines 255–271), described in more detail in the Methods (lines 669-681), and further\ndiscussed in the Discussion (lines 411–433) as well as in Figure 9. This ensures full transparency and\nimproves the interpretability of host immune responses under both dietary conditions.\nFigure 3. Presenting the full time series data on egg shedding shown in the supplementary figure and\nexplaining the observed variation between groups while fed the same diet (i.e., days 21-49) would be more\ntransparent. Some of these differences appear significant and are not currently explained in the text.\nAdditionally, the methods or results should explain and show the specific statistical tests used to obtain the\nsignificance differences shown on the graph, and explain which specific timepoints, treatments, or\nindividuals are being compared. Currently, the meaning of these significance levels is unclear.\nA: The text of the revised manuscript has been revised as suggested. The full time series data previously\nshown in Supplementary Figure 3 have been incorporated into the main text (now Figure 3). The results are\nnow accompanied by a detailed description, including the statistical tests applied, explicit labeling of the\ncomparisons (groups and time points), and appropriate interpretation of the observed variation. This\nrevision ensures full transparency and clarifies the meaning of the significance levels displayed in the figure.\nAuthors` note – see explanation for these 3 comments below:\nR1 & 2 comment: Lines 211-213: These results do not seem to match the figure caption or reported statistics,\nwhich suggest that the made comparison is between diets, not within them. Reporting statistics\nin the main text would aid in clarity and support.\nFigure 4: There appears to be inaccuracy in the reporting or visualization of results in this figure.\nComparisons in 4A and 4B are unclear and should be indicated on the figure. Visually, figure 4B does not\nseem to show a statistically different effect of colonization in the A-diet versus the W-diet (the bars on the\ngraph appear identical), although the figure caption reports one. Figures 4C and 4D do not match the\nfindings reported in lines 211-216. These figures also show that each diet is more similar to itself, but do\nnot make within-diet comparisons.\nFigure 5: The results reported in the text (Lines 217-221) contradict the statistics presented in this figure\ncaption. While the effect sizes vary, colonization has statistically significant effects on the metabolome for\nboth diets, not just the Western diet. Additionally, to test whether H. diminuta alters the small intestinal\nbacteriome in a diet-dependent manner (lines 530-531) requires testing for an interaction between diet\nand colonization on these indices, which does not appear to have been done.\nA: We thank the reviewer for their detailed feedback regarding the results and visualization in Figures 4\n(now Figure 6) and 5 (now Figure 7). The inconsistencies arose during the final stages of manuscript\npreparation, when results were recalculated and additional analyses included shortly before submission,\nwhile the corresponding text and Methods were not fully updated. These discrepancies have now been\ncorrected in the major revision (see Results, lines 199–233, and Methods, lines 585–619). All statistical tests\nare now clearly reported in the main text, with explicit indication of the compared groups in both the text\nand figure legends. Ambiguities related to both figures have been clarified in the Results sections and figure\nlegends, and the interpretation of the comparisons is now explicitly stated.\n Lines 77-80: The relevance of therapeutics for H. diminuta is unclear. Negative impacts on humans\nor animals have not been described.\n Line 114: The statement that worms on the Western diet have a “typical morphology” and “normal\nlength” requires a clear, quantitative description of typical morphology.\n Lines 135-139: It’s essential to know the average total worm burden in each group at the end point\nof Experiment 1 in order to interpret this statement. Additionally, it is unclear whether these lines\nrefer to results from Experiment 1 or Experiment 2.\n Lines 144-145: The conclusion that “tapeworms markedly reduce their metabolic requirements” in\nresponse to a Western diet requires evidence not currently presented in the text.\n Lines 288-299: This is the first time that H. diminuta is described as a human pathogen. This comes\ntoo late in the manuscript to be a convincing broader extension of the work.\n Figure 3: This figure is difficult to interpret in its current form; it should highlight specific patterns or\ngenes of interest for greater clarity.\n Line 243-247: Fructose is the key factor driving separation between diet groups (according to line\n226). Yet in line 245, changes in the metabolome are discussed in the context of glucose, sucrose,\nand maltose. Providing some interpretation of the key fructose result would be helpful.\nA: All minor comments have been accepted and the manuscript revised accordingly.\nR3 comment: The authors present a well-designed study investigating how two distinct diets, one fiber-rich\nand the other representative of a Western dietary pattern, affect the development of the intestinal\ntapeworm Hymenolepis diminuta in a rat model. Their data clearly show that the Western-like diet slows\ndown development of this organism in a reversible manner. They hypothesize that the parasite gets into an\naestivation state due to factors related to the Western diet and the influence of this diet on the microbiota.\nThey support their hypothesis with gene expression and metabolomics data. These data are insightful. The\nmain finding is compelling and counterintuitive: not only does it suggest a hidden link between Western\nlifestyle and reduced tapeworm infections, but it also raises the possibility that tapeworms may evade\ndetection in modern societies due to their underdeveloped forms.\nA: We thank the reviewer for their thoughtful summary and encouraging feedback. We greatly appreciate\nthe recognition of our study design and findings, and we are pleased that the broader implications of our\nwork were clearly highlighted. This positive assessment is highly motivating for our future research.\nR3 comment: The limitation of the study is that, beyond Figure 2, the analyses become relatively\nsuperficial. The causal connections between tapeworm physiology and dietary components (or diet-derived\nmetabolites) are not definitively established and appear to fall outside the current scope of the paper.\nFurthermore, the study does not address how variations in diet composition might alter the outcome. For\ninstance, how would a modified Western diet formulated to reduce fructose levels in the gut impact the\nobserved effects? Fructose emerges as a potentially important metabolite in the analysis, yet its role\nremains speculative without experimental testing. Whether the study meets the threshold for publication\nin this journal without such deeper mechanistic or dietary exploration is ultimately an editorial decision.\nA: We thank the reviewer for this insightful and relevant comment, which rightly touches on the current\nscope of our study. To our knowledge, this is among the first studies to investigate the physiological\nresponse of H. diminuta to two contrasting host diets and to highlight the emergence of a previously\nunrecognized diet-associated developmental state. We see this work as the beginning of a new research\ndirection in helminth biology, which will need to be developed further in future studies and, as our findings\nsuggest, will not be straightforward.\nThe study was not designed to dissect causal mechanisms of individual dietary components, which would\nrequire a series of additional experiments—constituting a separate, more complex project. We agree,\nhowever, that fructose emerges from our data as a potentially interesting metabolite. Its role remains\nspeculative at this stage, as it has not been experimentally tested. To reflect this important point, we have\nrevised the Discussion (lines 395-402) and Conclusions (lines 454-456) to explicitly acknowledge fructose as\na candidate metabolite and to emphasize that targeted experiments focusing on fructose and other diet-\nderived metabolites represent a key direction for follow-up research.\nR3 comment: Minor comments:\n9\n Page 3, lines 93-97: Please clarify what the reported percentages refer to (mass percentage, caloric\ncontribution, etc.) [ad 1 response below]\n Page 7, lines 213-216: The conclusion that dietary fiber absence leads to \"destabilization\" of the\nmicrobiota is not fully supported by Figure 4. Please elaborate on how this conclusion is derived\nfrom the data. [ad 2 response below]\n Page 7, lines 237-239: The claim that specific diacylglycerols and amino acids are present only in\nanimals with H. diminuta is not directly supported by the cited figures. Please provide metabolite-\nlevel information or clarify the connection. [ad 3 response below]]\nA: We agree with the reviewer’s comments and have addressed all three points in the major revision.\n1. The composition of both diets has been corrected and clarified in Table 1, with values now explicitly\nexpressed as mass percentage of diet (% w/w) or per kg diet.\n2. The description of microbiota results has been revised in the Results and Discussion sections (lines\n199–233 and 353–384), where we now report reduced microbial richness and altered community\nstructure instead of using the less precise term “destabilization.”\n3. The metabolomic results have been clarified, with absolute metabolite-level data provided in the\ndedicated Figshare repository (https://figshare.com/s/df77505840944a777748 ). In the text, we\nnow describe these metabolites as “consistently enriched in colonized animals” rather than\n“present only.”\nREVIEWER #1 & #2: (REMARKS TO THE AUTHOR):\nMajor comments\nIntroduction and discussion:\nWe appreciate the additional context the revised manuscript provides in the\nintroduction about diet-microbiome relationships, but the existing literature on\nhelminth development and helminth-diet interactions, particularly as directly related to\nthis system, are still poorly represented. For example, lines 76-78 state that little is\nknown about the effects of host diet on helminths. This is not factual, there is a\nsubstantial amount of work linking diet with helminth infections. Therefore, more\nnuance is needed here to understand the precise gap this study is addressing. In\nparticular, line 404 of the discussion refers to a prior study in this system that found\nsimilar effects of diet on H. diminuta development as reported in this study, seemingly\ncontradicting statements throughout the paper that little is known about how diet\naffects helminths (lines 76-78), or whether helminths are capable of developmental\nplasticity (lines 279-280). In the context of helminth development, contrary to the\nauthors’ claims on lines 279-280, it is well-known that parasitic helminths can show\nflexibility in their developmental strategies (including hypobiosis and similar\nphenomena) and the authors need to clarify how their study builds on what is already\nknown. The strategy of moving all information about helminth development to the\ndiscussion (e.g., lines 272-278) only further detracts from having a transparent\ndiscussion of the literature this study is building upon up front in the introduction.\nCrucially, this information cannot be decoupled from the gap the authors are\nsuggesting exists in the helminth literature.\nA: We thank the reviewer for this very constructive comment, which indeed helped us\nrefine the conceptual coherence of the manuscript. We agree that our previous\nwording understated the existing body of work on helminth developmental plasticity\nand helminth–diet interactions. At the same time, we are constrained by the journal’s\nlimits (1000 words for the Introduction, 5000 words for the full manuscript, and a\nmaximum of 70 references). The current version is already close to all three limits,\nand due to the multidisciplinary scope of the study we have exceeded the reference\nlimit; we are now actively consolidating citations where possible.\nIn response to the reviewer’s concerns, we have substantially revised the\nrelevant sections to more accurately reflect the state of the literature:\n The original statement suggesting that “little is known about dietary effects on\nhelminths…” (former lines 76–78) has been rewritten to acknowledge existing\nstudies across different host–parasite systems, including diet-dependent shifts\nin helminth persistence and developmental arrest (now lines 76–82).\n We improved the contextual link between historical observations of nutritional\nsensitivity in Hymenolepis diminuta and the rationale for our study design\n(lines 107-108).\n We incorporated the relevant reference (Myhill et al., 2018, 2020; Carvalho et\nal., 2023).\n We further revised the passages in the Discussion referring to helminth\ndevelopmental flexibility (former lines 279–280), clarifying that plasticity —\nincluding hypobiosis — is already established in some helminth taxa and\nsituating our findings within this broader framework (now lines 282–284).\nThese revisions address the reviewer’s concerns while remaining within the journal’s\nstrict word and reference limits. We believe the updated text now provides a clearer\nand more accurate representation of the existing literature and how our study builds\nupon it.\nMethods:\nOverall, the methods are well laid-out and much clearer in the revised document. We\nappreciate the additional detail the authors provide about sample sizes and statistical\nanalyses, and the new visualization of the experimental design (Figure 1). However,\nwe still have some concerns about the authors’ presentation of their study design.\nFirst, we agree with the authors that acknowledging experiment 1 and experiment 2\ntest distinct phenomena is a good approach (that is, experiment 1 tests the effects of\na Western diet on development to the adult stage while experiment 2 tests the\nreversibility of this diet’s effects on reproduction when temporarily presented to adult\nworms). However, while the authors do well at acknowledging these two distinct\nphenomena in the discussion (lines 293-299), they need to be upfront about this\ndistinction in the methods and results section as well, rather than stating that this\nsecond experiment is a test of developmental reversibility, which is misleading (e.g.,\nlines 142-144).\nA: We agree with the reviewer’s point and have revised the Methods and Results\nsections to clearly distinguish between the two experimental aims (Results: lines\n123–125, 146–149; Methods: lines 505–506, 521–522).\nSecond, while we appreciate the clarification of how colonization was determined\n(lines 137-142), these are inconsistent methods for determine colonization depending\non the treatment group (egg shedding vs adult worm count at necropsy), which could\nimpact the interpretation of results. The authors need to candidly discuss how this\ninconsistency in measuring colonization may affect their interpretations.\nA: We thank the reviewer for this important point. We have clarified the\nmethodological differences in the Results (lines 133–139) and added a concise\ndiscussion of how egg-based detection versus necropsy confirmation may influence\ninterpretation (Discussion, lines 315–321). These additions improve transparency\nregarding the limitations of cross-diet comparisons.\nResults:\nWe continue to have concerns about the alignment between results presented in\nsome figures and results reported in the text. For example, the Simpson’s diversity\nresult presented on lines 212-214 is not supported by Figure 6B, and the figure\ncaption does not contain any additional information to clarify this discrepancy.\nA: We thank the reviewer for carefully checking the alignment between the text and\nfigures. We identified an error in the original script that resulted in an incorrect x-axis\nscale in Figure 6B. This has now been corrected.\nWe have clarified in both the text and the figure legend that alpha diversity was\ncalculated using Simpson’s index (1 − D), where values approaching 1 indicate\nhigher diversity. The revised Figure 6B now displays the correct 0–1 scale, and the\ncorresponding sections in the Results (lines 216-217), Discussion (line382), and\nMethods (line 625) have been updated. The corrected figure and text consistently\nshow higher microbial diversity in rats fed the Accessible Fiber diet.\nFurthermore, while the immune data we requested is now discussed in the main text,\nbased on the figure these results are not being represented accurately. For example,\nFigure 9 shows significant differences between colonized and non-colonized rats for\nIL-1beta, IFNg, and IL4, but the text states that there is no differences in any of these\ncytokines between the groups and goes on to interpret the results based on a\nseeming erroneous conclusion (lines 253-259). It is not clear to us whether the\nstatistical results/figures are incorrectly reported, whether the authors misinterpreted\nthe statistics in the text, or some other issue. However, this is a major issue that must\nbe addressed.\nA: We thank the reviewer for identifying this issue. We apologize for the error in the\noriginally reported textual description. After re-examining the dataset, we confirmed\nthat the underlying data and statistical analyses were correct, but the corresponding\ntext misrepresented the outcomes. This has now been fully corrected. The Results\nsection (lines 256–262) and Discussion section (lines 426–440) have been rewritten\nto accurately reflect the significant differences shown in Figure 9. We sincerely\nappreciate the reviewer’s careful attention and patience.\nMinor comments\nLine 66: Helminths have not “largely vanished from modern populations”.\nApproximately 2 billion people are infected with helminths globally. If this statement is\nmeant to refer only to Western nations, the authors should state this clearly.\nA: Has been corrected (line 66).\nLine 122: A quantitative definition/source for \"normal length\" in H. diminuta is needed\nhere.\nA: Has been incorporated (line 127).\nREVIEWER #3 (REMARKS TO THE AUTHOR):\nThe authors have addressed my previous minor concerns, and their major revision\nhas significantly clarified the scope of the manuscript. The reorganization of the\nmanuscript, including the revised introduction and the separation of the discussion\nsection, has also improved overall clarity. I have no additional general comments\nbeyond what I stated in my initial review.\nA: We thank the reviewer for the positive feedback. This is greatly appreciated.\nMinor comments:\n* In the revised and relocated Table 1, the main components of the Western diet are\nnot fully represented. Only butter fat and cholesterol are listed, whereas the original\nsupplementary table included several additional ingredients. Theis appears to be a\nmistake.\nA: We apologize for this oversight. The missing components have now been added\nto the revised Table 1.\n* The new discussion is helpful and more clearly framed, but it is quite long. I\nrecommend trimming redundant or lower-priority content to improve focus and\nreadability.\nA: We thank the reviewer for the recommendation. We have trimmed and refined the\ndiscussion to reduce redundancies (see tracked changes). However, more\nsubstantial cuts would compromise the interpretation of complex, multi-layered\ndatasets, so we propose retaining the current streamlined version. We appreciate the\nreviewer’s understanding."}]

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