Celastrol (Cel), derived from the traditional herb Tripterygium wilfordii Hook. f., has anti-inflammatory, anti-tumor, and immunoregulatory activities. Renal dysfunction, including acute renal failure, has been reported in patients following the administration of Cel-relative medications. However, the functional mechanism of nephrotoxicity caused by Cel is unknown. This study featured combined use of activity-based protein profiling and metabolomics analysis to distinguish the targets of the nephrotoxic effects of Cel. Results suggest that Cel may bind directly to several critical enzymes participating in metabolism and mitochondrial functions. These enzymes include voltage-dependent anion-selective channel protein 1 (essential for maintaining mitochondrial configurational and functional stability), pyruvate carboxylase (involved in sugar isomerization and the tricarboxylic acid cycle), fatty acid synthase (related to beta-oxidation of fatty acids), and pyruvate kinase M2 (associated with aerobic respiration). Proteomics and metabolomics analysis confirmed that Cel-targeted proteins disrupt some metabolic biosynthetic processes and promote mitochondrial dysfunction. Ultimately, Cel aggravated kidney cell apoptosis. These cumulative results deliver an insight into the potential mechanisms of Cel-caused nephrotoxicity. They may also facilitate development of antagonistic drugs to mitigate the harmful effects of Cel on the kidneys and improve its clinical applications.

The advanced single-cell RNA sequencing (scRNA-seq) technology enables the measurement of gene expression levels and identification of cell-specific markers in individual cells, and the revealing of cell types, providing valuable insights into cell behavior. ScRNA-seq has emerged as an effective method for detecting rare cell populations, cellular heterogeneity, and cell-to-cell crosstalk under physiology and pathology conditions, thereby advancing the revolution of toxicology research. In this review, we first briefly introduce the concept of hepatotoxicity and nephrotoxicity, along with their underlying mechanisms. We then summarize the single-cell sequencing technology, with an emphasis on single-cell isolation techniques, particularly for the most used droplet-based methods. Finally, we review the recent advances in the application of single-cell RNA sequencing in studying hepatotoxicity and nephrotoxicity. Overall, this review provides a comprehensive understanding of the recent advances in hepatotoxicity and nephrotoxicity studies at a single-cell resolution.

Hepatocellular carcinoma (HCC) is among the deadliest malignancies worldwide and still a pressing clinical problem. Icaritin, a natural compound obtained from the Epimedium genus plant, has garnered significant attention as a potential therapeutic drug for HCC therapies. Mitophagy plays a crucial role in mitochondrial quality control through efficiently eliminating damaged mitochondria. However, the specific mechanisms of the interplay between mitophagy and apoptosis in HCC is still unclear. We aimed to explore the cross-talk between icaritin-induced mitophagy and apoptosis in HCC cells and investigate its potential mechanisms. Firstly, we confirmed that icaritin inhibits proliferation and migration while inducing mitochondrial damage and reactive oxygen species (ROS) production in HCC cells. Secondly, based on proteomics analysis, we discovered that icaritin inhibits the growth of tumor cells and disrupts their mitochondrial homeostasis through the regulation of both mitophagy and apoptosis. Thirdly, icaritin causes mitophagy mediated by PINK1-Parkin signaling via regulating feedforward loop. Furthermore, knockdown of PINK1/Parkin leads to inhibition of mitophagy, which promotes cell death induced by icaritin in HCC cells. Finally, autophagy/mitophagy inhibitors remarkably enhance icaritin-induced cell death and anticancer efficacy. Collectively, our findings reveal that icaritin suppresses growth, proliferation and migration of HCC cell through induction of mitophagy and apoptosis, while inhibition of mitophagy significantly increased the anti-cancer and pro-apoptotic effects of icaritin, indicating that targeting autophagy or mitophagy is a novel approach to overcome drug resistance and enhance anticancer therapies.

Sepsis is a life-threatening syndrome leading to hemodynamic instability and potential organ dysfunction. Oridonin, commonly used in Traditional Chinese Medicine (TCM), exhibits significant anti-inflammation activity. To explore the protective mechanisms of oridonin against the pathophysiological changes, the authors conducted single-cell transcriptome (scRNA-seq) analysis on septic liver models induced by cecal ligation and puncture (CLP). They obtained a total of 63,486 cells, distributed across 11 major cell clusters, and concentrated their analysis on four specific clusters (hepatocytes/Heps, macrophages, endothelial/Endos and T/NK) based on their changes in proportion during sepsis and under oridonin treatment. Firstly, biological changes in Hep, which are related to metabolic dysregulation and pro-inflammatory signaling, are observed during sepsis. Secondly, they uncovered the dynamic profiles of macrophage's phenotype, indicating that a substantial number of macrophages exhibited a M1-skewed phenotype associated with pro-inflammatory characteristics in septic model. Thirdly, they detected an upregulation of both inflammatory cytokines and transcriptomic factor Nfkb1 expression within Endo, along with slight capillarization during sepsis. Moreover, excessive accumulation of cytotoxic NK led to an immune imbalance. Though, oridonin ameliorated inflammatory-related responses and improved the liver dysfunction in septic mice. This study provides fundamental evidence of the protective effects of oridonin against sepsis-induced cytokine storm.

Perfluorooctanoic acid (PFOA) is a man-made chemical broadly distributed in various ecological environment and human bodies, which poses potential health risks. Its toxicity, especially the male reproduction toxicity has drawn increasing attention due to declining birth rates in recent years. However, how PFOA induces male reproductive toxicity remains unclear. Here, we characterize PFOA-induced cell injury and reveal the underlying mechanism in mouse Leydig cells, which are critical to spermatogenesis in the testes. We show that PFOA induces cell injury as evidenced by reduced cell viability, cell morphology changes and apoptosis induction. RNA-sequencing analysis reveals that PFOA-induced cell injury is correlated with compromised autophagy and activated endoplasmic reticulum (ER) stress, two conserved biological processes required for regulating cellular homeostasis. Mechanistic analysis shows that PFOA inhibits autophagosomes formation, and activation of autophagy rescues PFOA-induced apoptosis. Additionally, PFOA activates ER stress, and pharmacological inhibition of ER stress attenuates PFOA-induced cell injury. Taken together, these results demonstrate that PFOA induces cell injury through inhibition of autophagosomes formation and induction of ER stress in Leydig cells. Thus, our study sheds light on the cellular mechanisms of PFOA-induced Leydig cell injury, which may be suggestive to human male reproductive health risk assessment and prevention from PFOA exposure-induced reproductive toxicity.

Tri (2-Ethylhexyl) phosphate (TEHP), widely used as a fire retardant and plasticizer, has been commonly found in the environment. Its potential health-related risks, especially reproductive toxicity, have aroused concern. However, the potential cellular mechanisms remain unexplored. In this study, we aimed to investigate the molecular mechanisms underlying TEHP-caused cell damage in Sertoli cells, which play a crucial role in supporting spermatogenesis. Our findings indicate that TEHP induces apoptosis in 15P-1 mouse Sertoli cells. Subsequently, we conducted RNA sequencing analyses, which suggested that ER stress, autophagy, and MAPK-related pathways may participate in TEHP-induced cytotoxicity. Furthermore, we demonstrated that TEHP triggers ER stress, activates p38 MAPK, and inhibits autophagy flux. Then, we showed that the inhibition of ER stress or p38 MAPK activation attenuates TEHP-induced apoptosis, while the inhibition of autophagy flux is responsible for TEHP-induced apoptosis. These results collectively reveal that TEHP induces ER stress, activates p38, and inhibits autophagy flux, ultimately leading to apoptosis in Sertoli cells. These shed light on the molecular mechanisms underlying TEHP-associated testicular toxicity.

Diabetic nephropathy (DN) is a severe complication of diabetes, characterized by changes in kidney structure and function. The natural product rosmarinic acid (RA) has demonstrated therapeutic effects, including anti-inflammation and anti-oxidative-stress, in renal damage or dysfunction. In this study, we characterized the heterogeneity of the cellular response in kidneys to DN-induced injury and RA treatment at single cell levels. Our results demonstrated that RA significantly alleviated renal tubular epithelial injury, particularly in the proximal tubular S1 segment and on glomerular epithelial cells known as podocytes, while attenuating the inflammatory response of macrophages, oxidative stress, and cytotoxicity of natural killer cells. These findings provide a comprehensive understanding of the mechanisms by which RA alleviates kidney damage, oxidative stress and inflammation, offering valuable guidance for the clinical application of RA in the treatment of DN.

Apoptosis as a common form of programmed cell death, plays a crucial role in tumor therapy. Celastrol and erianin are natural compounds extracted from plants. Celastrol exhibits cytotoxic effects on various tumor cells through mechanisms such as ferroptosis and apoptosis, while erianin significantly inhibits the growth and metastasis of cancer cells. However, the high toxicity of celastrol and the low water solubility of both compounds hinder their clinical applications. In this study, nanoparticles (CEN) self-assembled from celastrol and erianin were designed to enhance drug solubility and tumor-targeting capability. These nanoparticles significantly inhibited the proliferation of tumor cells while effectively killing the entire tumor cell population. Additionally, using activity-based protein profiling (ABPP) and a celastrol-probe (cel-p), Annexin A2 was identified as the target of celastrol in 4 T1 cells. Proteomics was subsequently used to evaluate the changes in protein expression induced by CEN. In a mouse model of breast cancer, CEN demonstrated superior tumor-targeting ability and prolonged action due to the enhanced permeability and retention (EPR) effect, resulting in better therapeutic efficacy with lower systemic toxicity. In conclusion, this study provides a novel reference for the treatment of breast cancer.

Tris (2-ethylhexyl) phosphate (TEHP) is a frequently used organophosphorus flame retardant with significant ecotoxicity and widespread human exposure. Recent research indicates that TEHP has reproductive toxicity. However, the precise cell mechanism is not enough understood. Here, by using testicular mesenchymal stromal TM3 cells as a model, we reveal that TEHP induces apoptosis. Then RNA sequencing analysis, immunofluorescence, and western blotting results show that THEP inhibits autophagy flux and enhances endoplasmic reticulum (ER) stress. Moreover, the activation of the ER stress is critical for TEHP-induced cell injury. Interestingly, TEHP-induced ER stress is contributed to autophagic flux inhibition. Furthermore, pharmacological inhibition of autophagy aggravates, and activation of autophagy attenuates TEHP-induced apoptosis. In summary, these findings indicate that TEHP triggers apoptosis in mouse TM3 cells through ER stress activation and autophagy flux inhibition, offering a new perspective on the mechanisms underlying TEHP-induced interstitial cytotoxicity in the mouse testis.

Tributyltin chloride (TBTCL) is a widely used fungicide and heat stabilizer in compositions of PVC. TBTCL has been detected in human bodies and potentially causes harmful effects on humans' thyroid, cardiovascular and other organs. As one of the first examples of endocrine disruptors, the toxicity effects of TBTCL on the male reproduction system have aroused concerns. However, the potential cellular mechanisms are not fully explored. In the current study, by using Sertoli cells, a critical regulator of spermatogenesis as a cell model, we showed that with 200 nM exposure for 24 h, TBTCL causes apoptosis and cell cycle arrest. RNA sequencing analyses suggested that TBTCL probably activates endoplasmic reticulum (ER) stress, and disrupts autophagy. Biochemical analysis showed that TBTCL indeed induces ER stress and the dysregulation of autophagy. Interestingly, activation of ER stress and inhibition of autophagy is responsible for TBTCL-induced apoptosis and cell cycle arrest. Our results thus uncovered a novel insight into the cellular mechanisms for TBTCL-induced toxicology in Sertoli cells.

Sepsis is a difficult-to-treat systemic condition in which liver dysfunction acts as both regulator and target. However, the dynamic response of diverse intrahepatic cells to sepsis remains poorly characterized. Capsaicin (CAP), a multifunctional chemical derived from chilli peppers, has recently been shown to potentially possess anti-inflammatory effects, which is also one of the main approaches for drug discovery against sepsis. We performed single-cell RNA transcriptome sequencing on 86,830 intrahepatic cells isolated from normal mice, cecal ligation and puncture-induced sepsis model mice and CAP-treated mice. The transcriptional atlas of these cells revealed dynamic changes in hepatocytes, macrophages, neutrophils, and endothelial cells in response to sepsis. Among the extensive crosstalk across these major subtypes, KC_Cxcl10 shared strong potential interaction with other cells when responding to sepsis. CAP mitigated the severity of inflammation by partly reversing these pathophysiologic processes. Specific cell subpopulations in the liver act collectively to escalate inflammation, ultimately causing liver dysfunction. CAP displays its health-promoting function by ameliorating liver dysfunction induced by sepsis. Our study provides valuable insights into the pathophysiology of sepsis and suggestions for future therapeutic gain.

Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has been considered an emerging and potentially toxic pollutant in recent years. Long-term exposure to TCS has been suggested to exert endocrine disruption effects, and promote liver fibrogenesis and tumorigenesis. This study was aimed at clarifying the underlying cellular and molecular mechanisms of hepatotoxicity effect of TCS at the initiation stage. C57BL/6 mice were exposed to different dosages of TCS for 2 weeks and the organ toxicity was evaluated by various measurements including complete blood count, histological analysis and TCS quantification. Single cell RNA sequencing (scRNA-seq) was then carried out on TCS- or mock-treated mouse livers to delineate the TCS-induced hepatotoxicity. The acquired single-cell transcriptomic data were analyzed from different aspects including differential gene expression, transcription factor (TF) regulatory network, pseudotime trajectory, and cellular communication, to systematically dissect the molecular and cellular events after TCS exposure. To verify the TCS-induced liver fibrosis, the expression levels of key fibrogenic proteins were examined by Western blotting, immunofluorescence, Masson’s trichrome and Sirius red staining. In addition, normal hepatocyte cell MIHA and hepatic stellate cell LX-2 were used as in vitro cell models to experimentally validate the effects of TCS by immunological, proteomic and metabolomic technologies. We established a relatively short term TCS exposure murine model and found the TCS mainly accumulated in the liver. The scRNA-seq performed on the livers of the TCS-treated and control group profiled the gene expressions of > 76,000 cells belonging to 13 major cell types. Among these types, hepatocytes and hepatic stellate cells (HSCs) were significantly increased in TCS-treated group. We found that TCS promoted fibrosis-associated proliferation of hepatocytes, in which Gata2 and Mef2c are the key driving TFs. Our data also suggested that TCS induced the proliferation and activation of HSCs, which was experimentally verified in both liver tissue and cell model. In addition, other changes including the dysfunction and capillarization of endothelial cells, an increase of fibrotic characteristics in B plasma cells, and M2 phenotype-skewing of macrophage cells, were also deduced from the scRNA-seq analysis, and these changes are likely to contribute to the progression of liver fibrosis. Lastly, the key differential ligand-receptor pairs involved in cellular communications were identified and we confirmed the role of GAS6_AXL interaction-mediated cellular communication in promoting liver fibrosis. TCS modulates the cellular activities and fates of several specific cell types (including hepatocytes, HSCs, endothelial cells, B cells, Kupffer cells and liver capsular macrophages) in the liver, and regulates the ligand-receptor interactions between these cells, thereby promoting the proliferation and activation of HSCs, leading to liver fibrosis. Overall, we provide the first comprehensive single-cell atlas of mouse livers in response to TCS and delineate the key cellular and molecular processes involved in TCS-induced hepatotoxicity and fibrosis.

Tributyltin chloride (TBTCL), a commonly used antiseptic substance, is commonly found in the environment. Human exposure to TBTCL through the consumption of contaminated seafood, fish, or drinking water has aroused concern. It is well-characterized that TBTCL has multiple detrimental effects on the male reproductive system. However, the potential cellular mechanisms are not fully elucidated. Here, we characterized molecular mechanisms of TBTCL-induced cell injury in Leydig cells, a critical supporter for spermatogenesis. We showed that TBTCL induces apoptosis and cell cycle arrest in TM3 mouse Leydig cells. RNA sequencing analyses revealed that endoplasmic reticulum (ER) stress and autophagy were potentially involved in TBTCL-induced cytotoxicity. We further showed that TBTCL causes ER stress and inhibited autophagy flux. Notably, the inhibition of ER stress attenuates not only TBTCL-induces autophagy flux inhibition but also apoptosis and cell cycle arrest. Meanwhile, the activation of autophagy alleviates, and inhibition of autophagy exaggerates TBTCL-induced apoptosis and cell cycle arrest flux. These results suggest that TBTCL-induced ER stress and autophagy flux inhibition contributed to apoptosis and cell cycle arrest in Leydig cells, providing novel understanding into the mechanisms of TBTCL-induced testis toxicity.

There is great demand for the development of novel efficient therapeutic strategies or preventative measures to alleviate the life-threatening complications of type 2 diabetes. Hederasaponin C (PB5), a natural product, has been reported to exhibit significant therapeutic effects in various diseases; however, the possible effects and mechanism underlying PB5 in reducing diabetic renal complications have not been comprehensively reported. Here, we investigated the response of murine diabetic models to PB5 treatment using single-cell RNA-sequencing (scRNA-seq) and proteomics. Our findings revealed the dynamic transcriptional changes of renal cells in response to diabetic nephropathy. PB5 alleviated inflammatory injury by partially reducing pathophysiologic processes. In addition, we observed severe glomerular lesions and functional deficiencies, including GBM thickening and podocyte dysfunction, during the progression of diabetes, which were likewise attenuated by PB5. These results provide insight into how PB5 treatment improves diabetic symptoms and possibly serves as a novel protective measure and therapeutic strategy in the treatment of type 2 diabetes.

Ramie (Boehmeria nivea L.), belonging to Urticaceae, is principally used for fabric production. It is a well-known natural fiber material for ancient clothing. Despite its important position and application value, the understanding on genetic regulation mechanism of fiber quality is limited. Here, we generate a chromosome-scale, high-quality reference genome of ramie, in which, approximately 90.2% of the assembled sequences have been anchored to 14 pseudochromosomes. Totally 27,664 protein-coding genes are predicted which cover 268.24 Mb region of the genome. Comparative genomic analysis reveals that 2,047 and 796 gene clusters expand and contract, respectively, underlying significant genes in plant hormone signal transduction and cellulose/lignin biosynthesis pathways. An integrative analysis combining quantitative trait loci (QTL), comparative transcriptomic data, and cytological experiments unravels the molecular regulatory mechanism of ramie fiber fineness, especially the critical regulating role of ethylene. This study would lay a solid foundation for the research of molecular biology in ramie and provide valuable reference for the improvement of high-quality fiber varieties.

Unisexual taxa are commonly considered short-lived as the absence of meiotic recombination is supposed to accumulate deleterious mutations and hinder the creation of genetic diversity. However, the gynogenetic gibel carp ( Carassius gibelio ) with high genetic diversity and wide ecological distribution has outlived its predicted extinction time of a strict unisexual reproduction population. Unlike other unisexual vertebrates, males associated with supernumerary microchromosomes have been observed in gibel carp, which provides a unique system to explore the rationales underlying male occurrence in unisexual lineage and evolution of unisexual reproduction. Here, we identified a massively expanded satellite DNA cluster on microchromosomes of hexaploid gibel carp via comparing with the ancestral tetraploid crucian carp ( Carassius auratus ). Based on the satellite cluster, we developed a method for single chromosomal fluorescence microdissection and isolated three male-specific microchromosomes in a male metaphase cell. Genomic anatomy revealed that these male-specific microchromosomes contained homologous sequences of autosomes and abundant repetitive elements. Significantly, several potential male-specific genes with transcriptional activity were identified, among which four and five genes displayed male-specific and male-biased expression in gonads, respectively, during the developmental period of sex determination. Therefore, the male-specific microchromosomes resembling common features of sex chromosomes may be the main driving force for male occurrence in gynogenetic gibel carp, which sheds new light on the evolution of unisexual reproduction.

BACKGROUND:Water buffalo (Bubalus bubalis) are divided into river buffalo and swamp buffalo subspecies and are essential livestock for agriculture and the local economy. Studies on buffalo reproduction have primarily focused on optimal fertility and embryonic mortality. There is currently limited knowledge on buffalo embryonic development, especially during the preimplantation period. Assembly of the river buffalo genome offers a reference for omics studies and facilitates transcriptomic analysis of preimplantation embryo development (PED).METHODS:We revealed transcriptomic profile of four stages (2-cell, 8-cell, Morula and Blastocyst) of PED via RNA-seq (Illumina HiSeq4000). Each stage comprised three biological replicates. The data were analyzed according to the basic RNA-seq analysis process. Ingenuity analysis of cell lineage control, especially transcription factor (TF) regulatory networks, was also performed.RESULTS:A total of 21,519 expressed genes and 67,298 transcripts were predicted from approximately 81.94 Gb of raw data. Analysis of transcriptome-wide expression, gene coexpression networks, and differentially expressed genes (DEGs) allowed for the characterization of gene-specific expression levels and relationships for each stage. The expression patterns of TFs, such as POU5F1, TEAD4, CDX4 and GATAs, were elucidated across diverse time series; most TF expression levels were increased during the blastocyst stage, during which time cell differentiation is initiated. All of these TFs were involved in the composition of the regulatory networks that precisely specify cell fate. These findings offer a deeper understanding of PED at the transcriptional level in the river buffalo.

Sugarcane is an important industrial crop with a high sugar yield that has become a leading energy crop worldwide. It is widely cultivated in tropical and subtropical regions. Various diseases beset the cultivation of sugarcane. The molecular study of disease resistance in sugarcane is limited by its complicated genome. In our study, RNA-seq was employed to detect the mechanism of twisted leaf disease tolerance in modern cultivar sugarcane, which derived from Narenga porphyrocoma. We completed high-throughput transcriptomic sequencing of 12 samples, including three stages of a susceptible (NSBC1_T “H3–8”) and an unsusceptible cultivar (NSBC1_CK “H-19”) with two biological repeats, respectively. Using the Saccharum spontaneum genome as reference, the average mapping ratio of the clean data was over 70%. Among the differentially expressed genes between H3–8 and H3–19, we focused on the analysis of hormone pathways and resistance (R) genes. The results showed that twisted leaf disease triggers hormone networks and around 40% of R genes conditioned lower expression in the susceptible cultivar. One of the possible reasons for H3–8 being susceptible to twisted leaf disease might be the null/retarded response of R genes, especially in pre-onset stage (46% down-regulated) of pathogens infection.

Ramie, Boehmeria nivea (L.) Gaudich, family Urticaceae, is a plant native to eastern Asia, and one of the world's oldest fibre crops. It is also used as animal feed and for the phytoremediation of heavy metal‐contaminated farmlands. Thus, the genome sequence of ramie was determined to explore the molecular basis of its fibre quality, protein content and phytoremediation. For further understanding ramie genome, different paired‐end and mate‐pair libraries were combined to generate 134.31 Gb of raw DNA sequences using the Illumina whole‐genome shotgun sequencing approach. The highly heterozygous B. nivea genome was assembled using the Platanus Genome Assembler, which is an effective tool for the assembly of highly heterozygous genome sequences. The final length of the draft genome of this species was approximately 341.9 Mb (contig N50 = 22.62 kb, scaffold N50 = 1,126.36 kb). Based on ramie genome annotations, 30,237 protein‐coding genes were predicted, and the repetitive element content was 46.3%. The completeness of the final assembly was evaluated by benchmarking universal single‐copy orthologous genes (BUSCO); 90.5% of the 1,440 expected embryophytic genes were identified as complete, and 4.9% were identified as fragmented. Phylogenetic analysis based on single‐copy gene families and one‐to‐one orthologous genes placed ramie with mulberry and cannabis, within the clade of urticalean rosids. Genome information of ramie will be a valuable resource for the conservation of endangered Boehmeria species and for future studies on the biogeography and characteristic evolution of members of Urticaceae.

The blunt snout bream Megalobrama amblycephala is the economically most important cyprinid fish species. As an herbivore, it can be grown by eco-friendly and resource-conserving aquaculture. However, the large number of intermuscular bones in the trunk musculature is adverse to fish meat processing and consumption. As a first towards optimizing this aquatic livestock, we present a 1.116-Gb draft genome of M. amblycephala, with 779.54 Mb anchored on 24 linkage groups. Integrating spatiotemporal transcriptome analyses, we show that intermuscular bone is formed in the more basal teleosts by intramembranous ossification and may be involved in muscle contractibility and coordinating cellular events. Comparative analysis revealed that olfactory receptor genes, especially of the beta type, underwent an extensive expansion in herbivorous cyprinids, whereas the gene for the umami receptor T1R1 was specifically lost in M. amblycephala. The composition of gut microflora, which contributes to the herbivorous adaptation of M. amblycephala, was found to be similar to that of other herbivores. As a valuable resource for the improvement of M. amblycephala livestock, the draft genome sequence offers new insights into the development of intermuscular bone and herbivorous adaptation.