Abstract

   Maintaining a dynamic balance among various life-history traits is
   crucial for survival in a warming world, yet the underlying mechanisms
   remain enigmatic. In this study, we employed the western clawed frog
   (Xenopus tropicalis) as a model and conducted a long-tern experiment
   from zygotes to adult stage. We find that even within the previously
   considered normal temperature range, a 5 °C increase in ambient
   temperature can establish a new metabolic state, resulting in elevated
   oxidative stress and a shift in energy allocation towards immune
   defense at the expense of sexual development. This conceptual framework
   of temperature-dependent trade-off strategy suggests that, while some
   studies observed that warm temperature reduces the risk of infection,
   it is important to note that this change may present challenges in the
   form of accelerated aging and reduced fertility, especially in
   ectotherms. These results not only indicate a far more complex adaptive
   response to future climate change than previously anticipated, but also
   provide a concise method for constructing animal models to explore
   diseases related to homeostatic disorders.

   Subject terms: Zoology, RNA sequencing
     __________________________________________________________________

   A transcriptome atlas of Xenopus tropicalis reveals the role of the
   immune-reproductive trade-off mediated by metabolic reprogramming in
   maintaining homeostasis from the perspective of inter-organ crosstalk.

Introduction

   Global temperature has broken a new record
   ([54]https://wmo.int/media/news/july-sets-new-temperature-records) and
   is predicted to further increase by another 5 °C at the end of this
   century^[55]1. Global warming has posed a serious threat to
   biodiversity, which has increased interest in how organisms counter
   temperature changes. To maintain whole-body homeostasis and adapt to
   external conditions, the limited energy resources of organisms must be
   flexibly invested in growth, development, immunity, and reproduction
   according to external cues, requiring trade-offs among these
   life-history traits^[56]2. The outcomes of these trade-offs are often
   closely linked to individual fitness and population persistence.
   Therefore, a comprehensive understanding of the temperature-driven
   trade-off strategy is essential for predicting the effects of global
   warming and finding ways to improve physical health.

   However, previous studies have often yielded conflicting conclusions,
   due to potential noise factors (such as diet composition, ultraviolet
   light, humidity, predators, etc.) in natural systems and short
   experimental periods (from a few hours to months) in controlled
   experiments, as well as the limitation of subjects to a single organ or
   sex^[57]3–[58]5. Several questions can be raised. For example, what is
   the fundamental reason behind this paradox? What physiological traits
   are involved in temperature-driven trade-offs for adaption? How do they
   interfere with each other? What are the biological signals that trigger
   the trade-offs? Therefore, a conceptual framework elucidating the
   temperature-driven adaptive strategy is urgently needed.

   Amphibians are the most threatened vertebrates, with nearly 40% of
   species now at risk of extinction^[59]6. Previous studies have shown
   the acute and subacute effects of extreme temperatures on
   amphibians^[60]7–[61]9. However, global warming in nature is a slow and
   long-term process. Therefore, elucidating the adaptive plasticity of
   amphibians before environmental temperatures reach extremes is more
   useful for climate change risk assessment and management. Xenopus
   strains are the most geographically distributed amphibian taxa^[62]10,
   widely used in developmental biology, regenerative biology, and genetic
   research due to their phylogenetic importance and the fact that they
   possess 80% of human disease genes^[63]11–[64]14. Here, we used the
   western clawed frog (Xenopus tropicalis) as an example to investigate
   the temperature-driven trade-off strategy and elucidate the underlying
   mechanisms from an inter-organ perspective. First, to avoid confounding
   factors, we established animal models adapted to different temperatures
   (23 °C, 28 °C) in a controlled environment (Fig. [65]1A). Then, the
   vulnerability and resilience to ambient temperatures were measured on
   multiple organs at morphological, histological, gene expression and
   transcript alternative splicing levels, following by a comparative
   analysis with reptiles and mammals. Finally, we identified the
   signaling molecules that mediate inter-organ communication and
   illustrated how these signals regulate energy reallocation to achieve
   physiological trade-offs in response to temperature changes. We
   presented a conceptual framework that improves our understanding of the
   temperature-driven adaptive response. The dataset provided in this
   study will serve as a powerful resource for assessing climate change
   risks and designing appropriate conservation programs.

Fig. 1. Warm temperature induces an alteration of the energy metabolic state.

   [66]Fig. 1
   [67]Open in a new tab

   A Schematic diagram of subjected to a temperature reciprocal transfer
   experiment for 12 months starting at the zygote stage. B Schematic
   representation of the seven organ types for RNA sequencing. C Principal
   component analysis (PCA) based on gene expression. D Rose diagrams
   showing the numbers of DEGs between the Cool-acclimated and
   Warm-acclimated groups. The first ring (in light blue) refers to
   undetected transcripts for each organ the numbers in black represent
   the detected transcripts in each organ type. The second ring (in light
   green) refers to transcripts unregulated by temperature. The numbers in
   white present the DEGs significantly regulated by ambient temperature.
   The innermost ring refers to the number of DEGs in each organ type
   (dark green, downregulated; dark red, upregulated). *FDR adjusted
   p < 0.05, **<0.01, ***<0.001. Colored lines with asterisks indicate
   significantly differential pairs (purple, between Warm-acclimated and
   Cool-acclimated; red, Warm-acclimated involved; blue, Cool-acclimated
   involved). E Relative gene expression of intestinal markers in each
   temperature-treated group normalized to average expression of the
   Cool-acclimated groups (♂, male; ♀, female). Colored lines with
   asterisks indicate a significantly different pair. The data are given
   as mean ± SEM. F Heatmap showing the relative abundance (scaled FPKM)
   of hepatic DEGs between temperature-switched groups and
   temperature-acclimated groups, which are related to complement
   activation, macrophage chemotaxis, glycolysis/gluconeogenesis, fatty
   acid beta-oxidation, and amino acid degradation, and significantly
   expressed in at least one condition in male or female. The same color
   code is used in the entire figure. Significant differential gene
   expression was detected in at least one condition. G Partial metabolic
   network in liver for differently expressed genes related to glucose,
   fatty acid, and amino acid metabolism. Boxes from left to right
   represent the relative abundance of genes in Cool-acclimated,
   Cool-switched, Warm-switched, and Warm-acclimated groups, respectively.
   The upper boxes belong to males and the lower boxes belong to females.
   G6P glucose 6-phosphate, F6P fructose 6-bisphosphate, FBP fructose
   1,6-bisphosphate, PEP phosphoenolpyruvate, TCA cycle tricarboxylic acid
   cycle. H H&E staining of adipose tissue of frogs (♂, male; ♀, female)
   in different temperature-treated groups (scale bars, 200 μm). All
   experiments were conducted based on six biological replicates in each
   temperature-treated group with male:female = 3:3 (except Cool-switched
   group, which is 4:2).

Results

Warming reprograms energy metabolic states

   The phenotypic plasticity reflected by most organ weight was in
   accordance with the temperature-size rule (i.e., that ectotherms
   exposed to warm temperatures take shorter to achieve maturity at a
   smaller size than when exposed to cool temperatures^[68]15, especially
   for the organs with high metabolic rate (Fig. S[69]1A, Supplementary
   Data [70]1). The small intestine, playing a prominent role in energy
   intake and allocation, was the most sensitive one in both males and
   females. To fully assess the impact of ambient temperatures, we
   performed transcriptomic profiling of 168 samples derived from brain,
   heart, lung, liver, small intestine, muscle, and skin (Fig. [71]1B,
   Supplementary Data [72]2). Altogether, more than 7000 million reads
   were generated, and 23,239 genes (involving 23,824 transcripts) were
   identified from all samples after quality control (Fig. S[73]1B,
   Supplementary Data [74]1, [75]3). Principal component analysis showed
   that samples were clustered by organ types (Fig. [76]1C). This
   ontological dependence, reflected in the unbiased clustering, indicates
   the accuracy of our experimental protocols.

   In line with the organ weight pattern, global transcriptome comparison
   indicated the highest thermal responsiveness of digestive organs, and
   revealed that current temperature changes can mitigate the earlier
   effects to some extent. (Figs. [77]1D; S[78]1A, C; Supplementary
   Data [79]1, [80]2). The histomorphology results revealed that long-term
   warm-acclimation reduced the surface area and weakened the muscular
   layer of the small intestine (Fig. S[81]2A, B; Supplementary
   Data [82]1), effects that could attenuate digestion and absorption.
   After measurement of enteric cell-type-associated genes that reflect
   differentiation, microbial signals recognition, and physical barrier
   function^[83]16, we found that the expression of genes associated with
   stem cells (Lgr5), entero-endocrine system (Chga, Tph1, Gip), and
   enterocytes (Maf, Nr1h4) was promoted by warm temperatures
   (Fig. [84]1E, Supplementary Data [85]2). The rapid self-renewal of
   intestinal cells was further corroborated by the upregulation of Elf3
   and Klf5 (Fig. [86]1E, Supplementary Data [87]2), which is essential
   for the differentiation and proliferation of small intestinal
   epithelial cells^[88]17,[89]18. We detected mutually exclusive
   expression of Foxo1 being inhibited, but Cldn2 being induced by warm
   temperature in males. Foxo1 could enhance intestinal barrier function
   by regulating mucus secretion and tight junction proteins^[90]19. In
   contrast, Cldn2 would lead to barrier loss and increased antigenic
   permeation^[91]20. These results suggest that warm temperatures
   contribute to the rate of intestinal cell renewal and increase
   intestinal permeability. Overall, the above phenotypic and molecular
   evidences indicate new energy requirements derived in warm
   temperatures.

   This increased intestinal permeability could lead to higher levels of
   endotoxins (e.g., lipopolysaccharide derived from gut microbiota) that
   reach the liver through the portal blood flow, and activate liver
   complement and macrophages (Fig. [92]1F, Supplementary Data [93]2).
   Many genes involved in glycolysis and gluconeogenesis were upregulated
   in the warm-acclimated group to provide rapid bursts of energy for
   immunocyte activation and migration^[94]21 (Fig. [95]1F, Supplementary
   Data [96]2), whereas the expression of Ugp2 was downregulated,
   indicating less glycogen stored (Fig. [97]1G, Supplementary
   Data [98]2). With the reduced oxygen input reflected by less expressed
   hemoglobin genes in both lung and skin (Fig. S[99]2C, Supplementary
   Data [100]1), the lower expression of genes related to amino acid
   degradation (Asl, Nags, Otc, Gls2, Ass1, Aldh7a1, Fah), fatty acid
   beta-oxidation (Acat1, Acadsb, Hadhb, Slc27a2) and triglyceride
   synthesis pathway (Acly, Fasn, Acss2, Gpat4, Dgat2) weakened the urea
   cycle and tricarboxylic (TCA) cycle (Fig. [101]1F, G; Supplementary
   Data [102]2), indicating a decreased utilization of protein and lipid.
   We made histologic sections of adipose tissue and measured the sizes of
   the adipocytes to validate the finding at the molecular level
   (Fig. [103]1H, Supplementary Data [104]2). The result showed that
   smaller adipocytes were more prevalent in the groups with warm
   temperature at the late stage (Fig. S[105]2D, Supplementary
   Data [106]1). In addition, the expression Dgat2, the rate-limiting
   enzyme gene in triglyceride synthesis, was positively correlated with
   adipocyte size and adipose weight (Fig. S[107]2E, F; Supplementary
   Data [108]1). These results suggest that ambient temperature can affect
   X. tropicalis in terms of the preference for nutrient utilization and
   storage, i.e., warm temperature induces alteration of metabolic state
   from fatty acid oxidation to glycolysis.

Warming promotes immune defense and ROS stress, especially in males

   Warm temperature accelerates the metabolic rates of ectotherms due to
   the thermodynamics of the biochemical reactions^[109]22. In this study,
   we found that genes related to peroxidase (Mpo, Epx) and cytochrome
   (Cyba, Cybb) were highly expressed in warm-acclimated males compared
   with their counterparts in the Cool-acclimated group (Fig. [110]2A,
   Supplementary Data [111]2), and previous studies have elaborated that
   these genes are strongly interconnected with ROS generation^[112]23.
   The accumulation of ROS will cause oxidative damage to various
   biological molecules, combined with low-grade systemic inflammation and
   accelerate the aging process^[113]24, which has been demonstrated in
   mice and fish^[114]25,[115]26. Therefore, the increase of ROS
   generation and systemic inflammation induced by warm temperature in our
   study (Figs. [116]2A; S[117]3A; Supplementary Data [118]1, [119]2)
   provides a molecular explanation for the conjecture of Cayuela et al.
   that global warming promotes amphibian aging, particularly in
   males^[120]27. To minimize the damage from ROS, the expression of genes
   related to antioxidant enzymes (Cat, G6pd) and DNA repair (Ogg1) was
   significantly upregulated (Fig. S[121]3B, Supplementary
   Data [122]1)^[123]28–[124]30, suggesting that a higher cost in terms of
   reducing oxidative damage was required in the warm-acclimated group.

Fig. 2. Warming promotes ROS stress and defense response, especially in
males.

   [125]Fig. 2
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   A Expression of genes associated with reactive oxygen species
   biosynthesis in males and females (left, male; right, female). The
   histograms show the relative abundance in each temperature-treated
   group normalized to the average expression of the Cool-acclimated
   group. The data are given as mean ± SEM (|log2 FC| > 1, FDR-adjusted
   p-value < 0.05). B Warm temperature activates the immune response. The
   heatmap (top right) shows the results of GO enrichment analysis between
   each group. The color bar indicates the FDR-adjusted p-value. CA
   Cool-acclimated, CS Cool-switched, WS Warm-switched, WA
   Warm-acclimated. Chord diagrams (left and down right) show immune
   response-related DEGs that were significantly expressed between the
   warm-acclimated group and the cool-acclimated group (|log2 FC| > 1, FDR
   adjusted p-value < 0.05. left, male; right, female). The length of the
   brick for each gene corresponds to the sum of |log2 FC| in multiple
   organs. The length of the brick for each organ corresponds to the sum
   of |log2 FC| in all related DEGs. *FDR adjusted p < 0.05, **<0.01,
   ***<0.001. Colored lines are described as in FC | >1, FDR adjusted
   p-value < 0.05. left, male; right, female). The length of the brick for
   each gene corresponds to the sum of |log2 FC| in all related DEGs. *FDR
   adjusted p < 0.05, **<0.01, ***<0.001. Colored lines are described as
   in Fig. [127]1. All experiments were conducted based on six biological
   replicates in each temperature-treated group, with male:female = 3:3
   (except Cool-switched group, which is 4:2).

   The by-product ROS can not only serve as a signal to drive metabolic
   switch from oxidative phosphorylation to glycolysis, but also supports
   immune response and pathogen clearance^[128]31. When summarizing the
   results of Gene Ontology (GO) enrichment analyses, we found that the
   responses to ambient temperature are sex-specific (Fig. S[129]4,
   Supplementary Data [130]1). For males, terms enriched in multiple
   organs were associated with immunity (e.g., inflammation response,
   respiratory burst, innate immune response and regulation of cytokine
   production), while the effect of ambient temperature on females was
   primarily in terms of metabolism (e.g., small molecule metabolic
   process, carboxylic acid metabolic process, and alpha-amino acid
   metabolic process). Therefore, we hypothesized that sex hormones and
   related signals might be closely linked to temperature-driven
   plasticity.

Warming attenuates sexual dimorphism

   To assess the relationship between temperature and sexual dimorphism,
   we compared the number of DEGs between males and females in each
   temperature-treated group. We found that the number decreased sharply
   with the increasing temperature, especially in the liver (Fig. [131]3A,
   Supplementary Data [132]2). This indicates that warm temperatures
   attenuate sexual dimorphism in amphibians at the transcriptomic level.
   To further examine the variation of these sexually dimorphic DEGs
   affected by temperature, we defined the genes showing significantly
   different patterns between males and females in the Cool-acclimated
   group as male- and female-biased genes, and then checked their
   differential expression pattern in the warm-acclimated group. We found
   that the male-biased genes were downregulated in warm-acclimated male
   frogs, whereas the female-biased genes were upregulated. Conversely,
   the male-biased genes were upregulated in warm-acclimated females,
   whereas the female-biased genes were downregulated (Fig. [133]3B).

Fig. 3. Warm temperature attenuates sexual dimorphism.

   [134]Fig. 3
   [135]Open in a new tab

   A The numbers of DEGs between male and female in different
   temperature-treated groups among multiple organs. B Warm temperature
   feminizes gene expression in males but masculinizes gene expression in
   females. Volcano plot of hepatic sex-biased genes in the
   Cool-acclimated group (left). Differences in hepatic gene expression
   between Warm-acclimated males and Cool-acclimated males categorized by
   sex-biased expression in Cool-acclimated frogs (middle). Differences in
   hepatic gene expression between Warm-acclimated females and
   Cool-acclimated females categorized by sex-biased expression in
   Cool-acclimated frogs (right). Colors indicate male-biased (red),
   female-biased (green), and unbiased (gray) expressed genes. The dotted
   lines paralleling to x axis or y axis indicate |log2 FC| = 1 or
   −log[10]adjust p-value = −log[10] 0.05, respectively. CA
   Cool-acclimated, WA Warm-acclimated. C The top 5 hepatic KEGG pathways
   were significantly enriched between male and female in the
   Cool-acclimated group. And the enrichment analysis of these
   corresponding pathways between the indicated contrasts. *FDR adjusted
   p < 0.05, **<0.01, ***<0.001. (male and female in a specific
   temperature-treated group, left; males in different temperature-treated
   groups, middle; females in different temperature-treated groups, right;
   CA Cool-acclimated, CS Cool-switched, WS Warm-switched, WA
   Warm-acclimated). D Heatmap showing the hepatic expression of genes
   involved in the steroid biosynthesis pathway between
   temperature-switched groups and temperature-acclimated groups and
   significantly expressed at least one condition in male or female. Gene
   expression is shown as the z-score of FPKM (fragments per kilobase of
   transcript per million mapped reads). The numbers on the right indicate
   the log2 FC values between male and female in a specific
   temperature-treated group. Significant differential gene expression was
   detected in at least one condition. E Representative images of testis
   (unilateral) in each temperature treatment (scale bars, 1 mm). F The
   histogram indicates the weight and the line indicates the relative
   weight of the testis (testis weight versus body weight) in each
   temperature treatment. The data are given as mean ± SEM. G Venn plots
   show the shared DEGs between indicated contrasts across multiple organs
   in males and females. H Summary of the relationship between sex
   phenotype, intron retention, and ambient temperature across lizard,
   frog, and mouse. The data are presented as mean ± SEM. Colored lines
   are described as in Fig. [136]1. All experiments were conducted based
   on six biological replicates in each temperature-treated group with
   male:female = 3:3 (except Cool-switched group, which is 4:2).

   To examine the biological significance of these sex-biased DEGs
   influenced by temperatures, KEGG pathway enrichment analysis was
   conducted on profiles of liver, as it showed pronounced sexual
   dimorphism. The result showed that steroid biosynthesis, carbon
   metabolism, pentose phosphate pathway, glycolysis/gluconeogenesis, and
   terpenoid backbone biosynthesis were the most significantly enriched in
   the Cool-acclimated or cool-switched groups (Fig. [137]3C,
   Supplementary Data [138]2). The number of significantly enriched terms
   decreased with increasing temperature, and even no significant
   enrichment was detected between males and females in the
   warm-acclimated group. When comparing individuals of the same sex
   raised in different temperature environments, these sexually dimorphic
   DEGs-related pathways in the cool environment were also significantly
   enriched in males (Fig. [139]3C, Supplementary Data [140]2). It is
   suggested that ambient temperature may have a more critical effect on
   sex development in male frogs, because sex hormones (such as
   progesterone, androgen, and estrogen) belong to steroids. Next, when
   checking the expression of DEGs included in the steroid biosynthesis
   term, we found that, overall, the impact of warm temperature could lead
   to the expression of these genes upregulated in male but downregulated
   in females, and lessen the sexual dimorphic pattern after a life cycle
   exposure (Fig. [141]3D, Supplementary Data [142]2). To further
   determine the impact of the sex-biased gene expression attenuation on
   the development of secondary sexual characteristics, we measured testis
   weight and relative weight (testis weight/body weight) (Fig. [143]3E,
   F; Supplementary Data [144]2), which are widely considered as measures
   of reproductive investment and sperm competition^[145]32,[146]33. We
   observed that warm temperatures exerted profound effects on testis
   weight. The significant reduction of both testis weight and relative
   weight corroborated the hypothesis that warm temperature suppressed the
   reproductive activity of X. tropicalis.

   The Venn plots showed the overlap of DEGs affected by temperature
   across multiple organs (Figs. [147]3G; S[148]5A; [149]3E, F;
   Supplementary Data [150]1, [151]2). Among these, two demethylase
   protein genes, Jarid2 and kdm6b, have been reported as mediators of
   male-to-female sex reversal triggered by high temperature in reptiles
   (Alligator mississippiensis, Trachemys scripta, and Pogona vitticeps)
   in terms of expression level or intron retention (IR)^[152]34–[153]36.
   For T. scripta, Ge et al. validated that the expression of Kdm6b was
   dramatically increased during development and its inhibition by warm
   temperature would lead to male-to-female sex reversal^[154]35. As for
   P. vitticeps, although some contradictory results exist at the
   molecular level about the effects of temperature on the two genes’ IR
   pattern, it is certain that warm temperature poses consistent effect on
   these two genes and causes sex reversal from male to
   female^[155]34,[156]36 (Figs. S[157]5B;[158]3E, F; Supplementary
   Data [159]1). However, in endotherms such as mice, the abundance of
   these genes is stable to ambient temperature (Figs. S[160]5C;[161]3E,
   F; Supplementary Data [162]1). Surprisingly, in our results with X.
   tropicalis, the two genes responded significantly to ambient
   temperature but in opposite patterns (Fig. S[163]5A, D, E). We found
   that the expression level and IR ratio of Jarid2 were significantly
   induced by warm temperature but the corresponding characteristics of
   Kdm6b were significantly inhibited (Figs. S[164]5A, D, E; [165]3E, F;
   Supplementary Data [166]1, [167]2). To our knowledge, X. tropicalis is
   currently not considered to be a temperature-dependent sex
   determination species. Our study indicated a complex but important
   relationship between these two genes, ambient temperature, and sex
   determination among amphibians, reptiles, and mammals (Fig. [168]3H).

Cholesterol metabolites and secreted proteins mediate inter-organ
communication for warm-adaptation

   Given the high temperature-sensitivity and central role in life
   activities, we focused on the liver, which is the main synthesis organ
   of cholesterol (responsible for nearly 80% of total cholesterol) and
   its metabolites (e.g., steroid hormones and bile acids). Steroid
   hormones and bile acids are well-known signal molecules for energy
   metabolism, sex development, and immune response^[169]37. Our data
   demonstrated that multiple genes participating in cholesterol
   biosynthesis were expressed at a higher level in the livers of females
   than males, and most of these genes were upregulated after warm
   acclimation in males but downregulated in females compared with those
   in the Cool-acclimated group (Fig. S[170]6, Supplementary Data [171]1).
   As for the bile acid biosynthesis process, the related genes were
   significantly depressed in warm-acclimated males, which showed opposite
   trends to cholesterol biosynthesis-related genes (Fig. S[172]6,
   Supplementary Data [173]1). These results implied that overall, the
   cholesterol biosynthesis process was more active in females than males.
   To acclimate to warm temperatures, males devoted more cholesterol to
   synthesizing steroid rather than bile acid, which feminized the
   metabolic state of males (Figs. S[174]6; [175]3D; Supplementary
   Data [176]1, [177]2). This finding agreed with our analysis of the
   attenuation of sexually dimorphic differences induced by warm
   temperature.

   In detail, we observed that the expression of rate-limiting enzyme
   encoded genes of the classical pathway (Cyp7a1) and alternative pathway
   (Cyp27a1 and Cyp7b1) of bile acid biosynthesis were both significantly
   depressed by warm temperature (about 8 to 80 fold) (Fig. S[178]6,
   Supplementary Data [179]1). But in mice, cold exposure induced bile
   acid synthesis by upregulating Cyp7b1 in the alternative pathway, but
   did not significantly affect Cyp7a1 or Cyp27a1^[180]38. These results
   indicated that the bile acid biosynthetic process in X. tropicalis was
   more sensitive to ambient temperature than in mammals, because at least
   75% of the total bile acids are produced by the classical pathway under
   normal conditions^[181]39. The bile acids secreted by the liver serve
   as regulators of gene expression in central and peripheral organs via
   their cognate receptors^[182]40. Consistent with mammals^[183]41, the
   negative feedback of hepatic bile acids and the expression of bile acid
   receptors (Nr1h4) in the small intestine were observed in X. tropicalis
   (Fig. [184]5A, Supplementary Data [185]2). In the warm-acclimated
   group, the downregulation of bile acid production and upregulation of
   intestine Nr1h4 expression reduce the enterohepatic utilization of
   lipid but promote the utilization of glucose^[186]42,[187]43 (Fig.
   S[188]6, Supplementary Data [189]1). Moreover, we also found the role
   of bile acids in maintaining glucose homeostasis through negative
   feedback of regulating glucose metabolism between the liver and
   skeletal muscle (Fig. S[190]6), which is similar to mammals^[191]42.
   Combining the above results, our data suggest that regulating nutrient
   utilization preference and inter-organ crosstalk through bile acids may
   be one of the strategies to adapt to different temperatures in X.
   tropicalis (Fig. [192]4A, B).

Fig. 5. A conceptual framework depicting temperature-driven trade-offs.

   [193]Fig. 5
   [194]Open in a new tab

   Warm temperature reduces the intestinal absorption area, increases
   intestinal permeability, and drives the metabolic state shift from
   fatty acid oxidation to glycolysis. The metabolic state adapted to warm
   temperature drives more oxidative stress, resulting in immune
   activation and the requirement for antioxidation, which weakens the
   energetic investment for growth and sexual development. The ROS,
   cholesterol metabolites (e.g., bile acids and steroids), and secreted
   proteins (e.g., IGF family) serve as molecular signals mediating
   inter-organ communication to orchestrate the adaptive trade-off
   strategy.

Fig. 4. Cholesterol metabolites and secreted proteins play a key role in
inter-organ communication for warm acclimation.

   [195]Fig. 4
   [196]Open in a new tab

   A Partial metabolic network related to the enterohepatic circulation of
   bile acid. Gene expression is shown as the z-score of FPKM. Significant
   differential gene expression was detected in at least one condition B
   Cross-talk between liver, intestine, and skeletal muscle mediated by
   bile acid. C Generalized steps and schematic framework for
   identification of the inter-organ cross-talk mediated by secreted
   protein, and showing the liver-small intestine interaction as an
   example. D Distribution of significance score for all hepatic genes and
   secreted protein genes across all small intestinal gene expression.
   Insulin-like growth factor-related genes (indicated by arrow) were
   identified as top-ranked. E Heatmap of correlation between the
   expression of hepatic Igf1 and organ weight. *FDR adjusted p < 0.05,
   **<0.01, ***<0.001. F Distribution of significance score for all
   hepatic genes and secreted protein genes across all small intestinal
   gene expression. Insulin-like growth factor-related genes (indicated by
   arrow) were identified as top-ranked. All experiments were conducted
   based on six biological replicates in each temperature-treated group
   with male:female = 3:3 (except Cool-switched group, which is 4:2).

   Inter-organ crosstalk mediated by secreted proteins is another critical
   mechanism for maintaining homeostasis^[197]44. A total of 678 secreted
   protein genes were identified from our transcriptomic profile. The
   heatmap and hierarchical cluster analysis revealed that the expression
   of these genes was organ-specific, and 348 genes were regulated by
   ambient temperature (Fig. S[198]7A). In order to explore the importance
   of secreted proteins in mediating inter-organ crosstalk, we employed
   the Quantitative Endocrine Network Interaction Estimation (QENIE)
   bioinformatic framework to identify correlations between liver and the
   small intestine (Fig. [199]4C). We found that among the secreted
   proteins, the expression of insulin-like growth factor related genes
   (Igf1, Igf2, and Igfbp4) were highly correlated with gene expression in
   the small intestine (Fig. [200]4D; Supplementary Data [201]4). Igf1,
   mainly generated in the liver, mediates energetic metabolism and
   enhances systemic growth^[202]45. We found that the expression of
   hepatic igf1 was significantly inhibited by warm temperature and was
   significantly correlated with multiple organ weight, particularly with
   the small intestine (Figs. S[203]7B; [204]4E; Supplementary
   Data [205]1, [206]2). After performing KEGG enrichment analysis of
   genes whose expression levels were synergistically changed by the
   expression of hepatic igf1 (Supplementary Data [207]5), we found that
   pathways associated with carbohydrate metabolism, lipid metabolism,
   nucleotide metabolism, cell growth and death were significantly
   enriched (Fig. [208]4F). These results suggested that the hepatic
   insulin-like growth factors may serve as regulators coordinating
   multiple organs to support the trade-off strategy through reprogrammed
   metabolism.

Discussion

   Mapping the phenotypic and molecular response for thermal adaption
   across a whole organism is important for understanding the effects of
   global warming. Previous studies are limited to a few tissues, a single
   sex, a short time period, or are confounded by uncontrolled noise.
   Here, based on a high-quality transcriptome atlas of the tropical
   clawed frog, we identified a wealth of temperature-driven physiological
   changes within and across organs, including energetic metabolism,
   antioxidant response, immunity, sexual development, and growth. In
   addition, the common and species-specific adaptive responses were
   uncovered by comparative analysis among amphibians, reptiles, and
   mammals. These results highlight the role of physiological trade-offs
   regulated by coordination across multiple organs in maintaining
   homeostasis and environmental adaptation.

   There is already a consensus that climate change is leading to a
   decline in biodiversity. However, it seems paradoxical that warm
   temperatures have been observed to improve immune
   response^[209]46,[210]47. Previous studies have reported that warm
   temperatures can reduce the prevalence of infections in
   amphibians^[211]48,[212]49. However, we must be aware that the immune
   activation induced by warm temperatures comes at a high cost. On the
   one hand, we found that warmth induces a new metabolic state that
   produces more reactive oxygen species, causing oxidative stress with a
   low inflammatory response, which is the same in both endotherms and
   ectotherms^[213]49–[214]51 (Figs. [215]2A; S[216]3A). Oxidative stress
   is a driver of ageing^[217]49. This explains the shortened lifespan of
   amphibians in the context of a warming climate observed in the
   field^[218]27. On the other hand, immune function is highly
   energy-intensive and must be traded off against other life-history
   traits, such as gamete production. Previous studies have shown that
   immunity and testosterone levels are often negatively correlated in
   male^[219]50; an overactive immune system can make pregnancy difficult
   in female^[220]51. In this study, we found that both sex dimorphism and
   sex organ mass were significantly reduced in highly immune individuals
   who adapted to warm environments (Fig. [221]3). This suggests that the
   rising temperatures are causing individuals to sacrifice energy
   expenditure for reproduction to ensure current survival, which is
   detrimental to population persistence. Many previous studies have
   ignored this point due to the short exposure period and a few
   monitoring indices. Therefore, our findings highlight the importance of
   long-term experiments and an integrated view of the metabolism,
   immunity, growth, and reproduction of individuals and even the health
   of future generations when assessing the biological effects of climate
   change or developing methods for animal conservation.

   Dysregulation of homeostasis may lead to various diseases, such as
   metabolic syndrome and related disorders (characterized by a cluster of
   metabolic and hormonal disorders disrupting lipid and glucose
   metabolism pathways)^[222]52, systemic inflammatory disorders
   (characterized by dysregulated excessive innate immune
   responses)^[223]53, immunological infertility (infertility caused by
   immunological disorders or a hyperactive immune
   system)^[224]50,[225]51, etc. A growing body of evidence suggests that
   elucidating the mechanisms of inter-organ communication in disease and
   its mediators is critical to understanding the regulation of
   homeostasis and the biology of systemic diseases^[226]54. Western
   clawed frog is an important model for medical research^[227]13,[228]55.
   Our study discovered the molecular signals mediating the energy
   trade-offs across multiple organs, such as ROS, bile acids, and
   secreted protein Igf1 etc. ROS drives metabolic switch from oxidative
   phosphorylation to glycolysis to meet new energy metabolism
   requirement^[229]31. The lessened bile acids in a warm environment
   reduce lipid absorption^[230]42, induce the overgrowth and
   translocation of intestinal bacteria^[231]56, and mediate systemic
   energy metabolism and immune response through the receptor in distant
   organs^[232]42. Igf1, mediating systemic glucose metabolic
   homeostasis^[233]57 and supporting sexual development and
   reproduction^[234]58 as well as suppressing immune
   inflammation^[235]59, is highly expressed in a cool environment.
   Therefore, our results not only provide clues to reveal the nature of
   amphibian population declines, but also provide a simple and
   inexpensive method of constructing animal models to explore the
   above-mentioned diseases. Meanwhile, our results also indicate the
   necessity of controlling the ambient temperature of experimental
   animals in medical research. Western clawed frogs are very sensitive to
   temperature, and the ambient temperatures can significantly affect
   various physiological functions of them, even within the range
   considered normal (23–28 °C)^[236]9. Therefore, in medical research,
   special attention should be paid to the stability of the ambient
   temperature to avoid the background noise interfering with the results
   of physiological experiments.

   In this study, using Xenopus tropicalis as an example, we present a
   conceptual framework for the physiological trade-offs caused by
   temperature changes (Fig. [237]5). Long-term warming established a new
   metabolic state, which allocated more energetic resources towards
   immune defense, but at the cost of reduced sexual dimorphism. Reactive
   oxygen species, cholesterol metabolites, and hepatic secreted proteins
   were the key signals mediating the defense-reproduction trade-offs
   through inter-organ communication. These results explain the molecular
   mechanisms of amphibians population decline under the scenario of
   global warming, emphasize the importance of considering the synergies
   and physiological trade-offs across multiple organs in climate change
   risk assessment, and also provide models and potential regulatory
   targets for elucidating the mechanisms underlying complex diseases
   (such as metabolic syndrome and immune infertility). Future studies
   will be carried out at different developmental stages (such as larval,
   metamorphic, and froglet stages) and at the single-cell RNA-sequencing
   scale to provide more detailed information.

Materials and methods

Animal rearing experiment

   A pair of adult X. tropicalis parents were purchased from Zhejiang
   University and were acclimated to room temperature with a photoperiod
   of 12:12 h. The frogs were fed three times daily with fish diet (JBL,
   Germany; raw protein: 43%; raw fat: 2.5%; raw fiber: 7%; ash: 7%;
   vitamin A: 25,000 IU kg^–1; vitamin D3: 2000 IU kg^–1; vitamin E:
   330 mg kg^–1; vitamin C: 400 mg kg^–1). The experimental protocols were
   approved by the Institution of Animal Care and the Ethics Committee of
   Chengdu Institute of Biology, Chinese Academy of Sciences
   (CIBDWLL2022007). We have complied with all relevant ethical
   regulations for animal use.

   To reduce the potential interference of host genotypic variation, all
   individuals surveyed in our study were the offspring of this couple of
   X. tropicalis parents. Breeding was induced using human chorionic
   gonadotropin. Adult X. tropicalis were injected with a dose of 20 U.
   Twenty-four hours later, the male and female were injected with doses
   of 150 U and 200 U, respectively, and transferred to a breeding
   chamber.

   All fertilized eggs were collected immediately and then randomly
   distributed into tanks containing laboratory-prepared water that was
   then placed in artificial climate boxes set to 23 °C or 28 °C (±1 °C,
   with humidity = 90% and a 12 h:12 h light/dark cycle). The temperatures
   were selected according to the optimal temperature range for X.
   tropicalis development as suggested by Khokha et al. ^[238]9, and
   temperatures outside of the above range will result in rapid death of
   the fertilized egg during cleavage stages. Animals were fed with
   pellets of fish diet (JBL), and the grinding level was adjusted to the
   size of their jaws. To ensure water quality (pH = 7.31–8.01, nitrite
   <1.0 mg L^–1, total NH4^+ <1 mg L^–1), aquaculture water was aerated
   for more than 24 h before use and was renewed every day, combined with
   tank cleaning. For the sake of even breeding density and avoiding
   crowding stress, some tadpoles were removed from the tanks as they
   grew.

   To investigate the response of X. tropicalis to temperature change, a
   reciprocal transfer experiment was performed between cool and warm
   environments when all animals within a group completed metamorphosis.
   Half of the froglets in cool and warm environments were transferred to
   their counterpart environment, and half remained in their original
   environment. Individuals that switched environments were considered as
   “temperature-switched” groups (warm-switched: Cool–Warm, cool-switched:
   Warm–Cool), whereas others that remained in their original environment
   were considered as “temperature-acclimated” groups (Cool-acclimated:
   Cool–Cool, warm-acclimated: Warm–Warm). Triplicate tanks were randomly
   used in each temperature treatment. The whole experiment lasted for 1
   year (Fig. [239]1A).

Sample collection

   At the end of the experiment, frogs were euthanized by MS-222
   (3-aminobenzoic acid ethyl ester methane sulfonate). The weights of
   representative organs were measured, including the brain, heart, lung,
   liver, stomach, small intestine, colon, kidney, adipose, femur, and
   testis. The gastrointestinal contents were removed before weighing.
   After measuring the entire length of the small intestine, the adipose
   tissue and the forepart of the jejunum were stored in 4%
   paraformaldehyde (Sigma) for light microscopy. Then, the results of the
   small intestine samples were suspended in RNA buffer (ThermoFisher
   Scientific) and stored at −80 °C, along with the brain, heart, lung,
   liver, skeletal muscle, and the skin of the back for RNA sequencing. A
   single animal is regarded as an experimental unit. All experiments were
   conducted based on six biological replicates in each
   temperature-treated group with male:female = 3:3 (except Cool-switched
   group, which is 4:2). A total of 24 animals participated in the
   experiment.

Histology

   After being weighed, the adipose tissue and the forepart of the jejunum
   were fixed by 4% paraformaldehyde (Sigma), paraffin-embedded, cut into
   5 μm-thick sections, and stained with hematoxylin-eosin using standard
   protocols. Briefly, the sections were dried on glass microscope slides,
   deparaffinized in xylene, and then hydrated in a graded alcohol series
   (100%, 100%, 95%, 90%, 80%, 70%) and washed with distilled water. Then,
   the sections were incubated in hematoxylin, washed with running tap
   water, differentiated in 1% acid alcohol, and again washed with running
   tap water. After being counterstained with eosin, the sections were
   dehydrated in a graded alcohol series (80%, 80%, 90%, 90%, 100%) and
   xylene. Lastly, the sections were mounted with neutral gum. Images were
   obtained using a digital three-lens camera microscope (BA400Digital).
   The pictures were analyzed using Fiji. The perimeter, villus length,
   and wall thickness of the small intestine were measured along with the
   area of adipocytes.

Transcriptome library preparation and sequencing

   Total RNA was isolated from the brain, heart, lung, liver, small
   intestine, skeletal muscle, and skin. RNA quality was validated with a
   BioAnalyzer (all samples had RIN >8). mRNA was purified from total RNA
   using poly-A oligo-attached magnetic beads. Sequencing libraries were
   generated using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina®
   (#E7530L, NEB, USA) according to the manufacturer’s recommendations.
   After internal size assessment, the libraries were sequenced on an
   Illumina platform, and 150 bp paired-end reads were generated.

Gene expression profiling

   After quality control and adaptor trimming using fastp software
   (version 0.23.1)^[240]60, the reads were mapped to the X. tropicalis
   genome (version 10.0) with HISAT2 software (version 2.1.0)^[241]61.
   Read counts for each gene in each sample were performed by HTSeq
   (version 0.6.0)^[242]62, and FPKM (Fragments Per Kilobase Million
   Mapped Reads) was then calculated to estimate the expression levels of
   genes in each sample. DESeq2 (1.20.0)^[243]63 was used to identify
   genes with differential expression in different temperature-treated
   groups. Genes with |log[2] fold-change| > 1 and p-adj < 0.05 were
   identified as differentially expressed genes (DEGs).

Function and pathway annotation

   The GO and KEGG (Kyoto Encyclopedia of Genes and Genomes,
   [244]http://kobas.cbi.pku.edu.cn/)^[245]64 enrichment of DEGs was
   implemented by the hypergeometric test, in which the p-value is
   calculated and adjusted by multiple comparisons as a q-value (genes in
   the whole genome were used as a data background when performing
   enrichment analysis). The terms with q < 0.05 were considered to be
   significantly enriched.

Intron retention analysis

   To compare the expression and splicing pattern of Jarid2 and Kdm6b
   across amphibian, reptile, and mammalian, we downloaded public data of
   P. vitticeps (NCBI with accession numbers PRJEB5206 and PRJNA699086)
   and M. musculus (NCBI with accession number PRJNA813534) treated by
   different temperatures. After quality control and adaptor trimming as
   mentioned above, sequences of each species were aligned to their genome
   using STAR software (v2.7.0f)^[246]65 with splice-aware alignment,
   refer to the accompanying Ensembl gene annotation (X. tropicalis:
   UCB_Xtro_10.0.107,
   [247]https://www.xenbase.org/xenbase/static-xenbase/ftpDatafiles.jsp;
   P. vitticeps: pvi1.1.100,
   [248]https://www.ncbi.nlm.nih.gov/assembly/GCF_900067755.1/; M.
   musculus: GRCm39.107,
   [249]https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.27/).
   Parameters are set to output only unique alignments.

   Differentially retained introns were detected in each species under
   different temperatures in order to conduct an unbiased analysis of
   splicing patterns. The IRFinder (v3.1) software^[250]66 was utilized to
   analyze the sequence library, generating a list of intron retention
   intervals with corresponding information. Candidate intron retention
   intervals were filtered based on the following criteria: intron
   retention ratio >0.5, interval coverage >0.9, and intron depth >1.0.
   Intron retention intervals were identified in the genes Jarid2 and
   Kdm6b across the three species. Intron retention ratios of each sample
   from the analysis results were displayed in bar plots and reported as
   mean ± SEM.

Inter-organ crosstalk regulator identification

   The communication between two organs varies in a coordinated manner.
   Differences in the expression of a secreted protein in one organ are
   associated with response genes in the target organ. To identify the
   secreted protein genes, the bicorrelation coefficient and corresponding
   p-value were calculated for all secreted and response gene pairs with
   respect to different temperatures using the bicorAndPvalue function
   from the WGCNA^[251]67 package. P-value matrices for secreted protein
   genes were ranked based on the significance level of association with
   the target organ, which was determined by the sum of the significance
   levels of all response genes. A filtering of the ranking results was
   performed to ensure that the secreted genes were primarily expressed by
   the origin organ. To analyze the regulatory role of a secreted protein
   gene, target genes with bicorrelation coefficients >0.5 and a
   p-value < 0.05 were selected for KEGG pathway and gene pathway
   enrichment analysis^[252]64.

Statistics and reproducibility

   The data were presented as mean ± SEM unless otherwise noted. The means
   of morphological and histological characteristics between groups were
   compared using one-way analysis of variance (ANOVA), assuming equal
   variance. The cool- or warm-acclimated groups were used as benchmarks
   for comparison with the other three groups (i.e., Warm–acclimated vs.
   Cool–acclimated; Cool-switched vs. Cool–acclimated, Warm-switched vs.
   Cool–acclimated, Warm-switched vs. Warm–acclimated, and Cool-switched
   vs. Warm–acclimated). All experiments were conducted based on six
   biological replicates in each temperature-treated group with
   male:female = 3:3 (except Cool-switched group, which is 4:2). A
   two-tailed Student’s t test was performed in R software (version 4.2.0)
   to calculate the p-value. The Holm–Bonferroni method was applied to
   correct the p-value with false discovery rate, and an adjusted p < 0.05
   was considered statistically significant (*, **, and *** indicate
   p < 0.05, p < 0.01, and p < 0.001, respectively).

Reporting summary

   Further information on research design is available in the [253]Nature
   Portfolio Reporting Summary linked to this article.

Supplementary information

   [254]Supplementary Information^ (15.2MB, pdf)
   [255]Supplementary Data 1^ (204.1KB, xlsx)
   [256]Supplementary Data 2^ (261.1KB, xlsx)
   [257]Supplementary Data 3^ (21.8KB, xlsx)
   [258]Supplementary Data 4^ (143.5KB, xls)
   [259]Supplementary Data 5^ (744.5KB, xls)
   [260]42003_2025_8340_MOESM7_ESM.docx^ (19.8KB, docx)

   Description of Additional Supplementary Files
   [261]Reporting Summary^ (190.5KB, pdf)

Acknowledgments