Evaluation of bromocriptine and plumbagin against the monogenean Rhabdosynochus viridisi: Computational drug repositioning and in vitro approaches.

Monogeneans are parasitic platyhelminths that can harm the health of farmed fish. Few treatments are available against monogeneans, and the incentive to develop new antiparasitic agents is similar or even lower than the incentive for neglected parasitic diseases in humans. Considering that searching for and developing new antimonogenean compounds may require enormous investments of time, money, and animal sacrifice, the use of a computer-guided drug repositioning approach is a reasonable alternative. Under this context, this study aimed to evaluate the effectiveness of plumbagin and bromocriptine against adults and eggs of the monogenean Rhabdosynochus viridisi (Diplectanidae). Plumbagin is a phytochemical compound that has recently emerged as a potent antimonogenean; however, further investigation is required to determine its effects on different monogenean species. Bromocriptine was selected through a computational approach that included molecular docking analyses of 77 receptors of monogeneans (putative drug targets) and 77 ligands (putative inhibitors). In vitro experiments showed that bromocriptine does not exhibit mortality at concentrations of 0.1, 1, and 10 mg/L whereas plumbagin at 2 and 10 mg/L caused 100% monogenean mortality after 3 h and 30 min, respectively. The most effective concentration of plumbagin (10 mg/L) did not completely inhibit egg hatching. These findings underscore plumbagin as a highly effective agent against adult monogeneans and highlight the need for research to evaluate its effect(s) on fish. Although computational drug repositioning is useful for selecting candidates for experimental testing, it does not guarantee success due to the complexity of biological interactions, as observed here with bromocriptine. Therefore, it is crucial to examine the various compounds proposed by this method.

Gestational diabetes mellitus: genetic factors, epigenetic alterations, and microbial composition.

Gestational diabetes mellitus (GDM) is a common metabolic disorder, usually diagnosed during the third trimester of pregnancy that usually disappears after delivery. In GDM, the excess of glucose, fatty acids, and amino acids results in foetuses large for gestational age. Hyperglycaemia and insulin resistance accelerate the metabolism, raising the oxygen demand, and creating chronic hypoxia and inflammation. Women who experienced GDM and their offspring are at risk of developing type-2 diabetes, obesity, and other metabolic or cardiovascular conditions later in life. Genetic factors may predispose the development of GDM; however, they do not account for all GDM cases; lifestyle and diet also play important roles in GDM development by modulating epigenetic signatures and the body's microbial composition; therefore, this is a condition with a complex, multifactorial aetiology. In this context, we revised published reports describing GDM-associated single-nucleotide polymorphisms (SNPs), DNA methylation and microRNA expression in different tissues (such as placenta, umbilical cord, adipose tissue, and peripheral blood), and microbial composition in the gut, oral cavity, and vagina from pregnant women with GDM, as well as the bacterial composition of the offspring. Altogether, these reports indicate that a number of SNPs are associated to GDM phenotypes and may predispose the development of the disease. However, extrinsic factors (lifestyle, nutrition) modulate, through epigenetic mechanisms, the risk of developing the disease, and some association exists between the microbial composition with GDM in an organ-specific manner. Genes, epigenetic signatures, and microbiota could be transferred to the offspring, increasing the possibility of developing chronic degenerative conditions through postnatal life.

Global DNA methylation and miR-126-3p expression in Mexican women with gestational diabetes mellitus: a pilot study.

BACKGROUND: Gestational diabetes mellitus (GDM), a type of diabetes that occurs for the first time during pregnancy, may predispose the development of chronic degenerative diseases and metabolic alterations in mother and offspring. DNA methylation and microRNA (miRNA) expression are regulatory mechanisms of gene expression that may contribute to the pathogenesis of GDM. Therefore, we determined global DNA methylation and miR-126-3p expression levels in 8 and 7 Mexican women with and without GDM, respectively. METHODS AND RESULTS: Global DNA methylation was assessed by measuring the percentage of 5-methylcytosine (5-mC) in placenta, umbilical cord, and plasma DNA samples, whereas miR-126-3p expression was quantified by real-time PCR using the 2-ΔCt method of the corresponding RNA samples. A significant increase in the percentage of 5-mC was detected in placenta samples from GDM patients compared to healthy women, while plasma samples showed a significant decrease. Conversely, miR-126-3p expression levels were significantly higher in plasma from the GDM group, while placenta and umbilical cord samples showed no significant differences across experimental groups. Furthermore, DNA methylation correlated significantly with glucose levels in placenta and plasma. Likewise, miR-126-3p expression correlated significantly with plasma glucose, in addition to maternal body mass index (BMI at first trimester). CONCLUSION: The results indicate that GDM is associated with alterations in global DNA methylation levels and miR-126-3p expression in placenta and/or plasma, providing insights into future novel approaches to diagnose and/or prevent this pathology.

Transcriptomic signaling in zebrafish (Danio rerio) embryos exposed to environmental concentrations of glyphosate.

Glyphosate [N-(phosphonomethyl)glycine] is one of the most popular herbicides worldwide. Globally, the use of glyphosate is increasing, and its residues have been found in drinking water and food products. The data regarding the possible toxic effects of this herbicide are controversial. Therefore, the aim of this study was to evaluate the effects of glyphosate at environmental concentrations in zebrafish (Danio rerio) embryos. Embryos were exposed to 0, 1, 100, and 1,000 µg/L glyphosate for 96 h, and mortality, heart rate, and hatching rate were evaluated. After the experiment, RNA was extracted from the embryos for transcriptional analysis. No mortality was recorded, and exposure to 100 µg/L and 1,000 µg/L of glyphosate resulted in lower heart rates at 48 h. In addition, RNA-seq analysis revealed that glyphosate exposure induced subtle changes in gene transcription profiles. We found 30 differentially expressed genes; however, the highest glyphosate concentration (1,000 µg/L) induced the greatest number of differentially expressed genes involved in oocyte maturation, metabolic processes, histone deacetylation, and nervous system development.

Global m6A RNA Methylation in SARS-CoV-2 Positive Nasopharyngeal Samples in a Mexican Population: A First Approximation Study.

The Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) is the causal agent of COVID-19 (Coronavirus Disease-19). Both mutation and/or recombination events in the SARS-CoV-2 genome have resulted in variants that differ in transmissibility and severity. Furthermore, RNA methylation of the N6 position of adenosine (m6A) is known to be altered in cells infected with SARS-CoV-2. However, it is not known whether this epitranscriptomic modification differs across individuals dependent on the presence of infection with distinct SARS-CoV-2 variants, the viral load, or the vaccination status. To address this issue, we selected RNAs (n = 60) from SARS-CoV-2 sequenced nasopharyngeal samples (n = 404) of 30- to 60-year-old outpatients or hospitalized individuals from the city of Mazatlán (Mexico) between February 2021 and March 2022. Control samples were non-infected individuals (n = 10). SARS-CoV-2 was determined with real-time PCR, viral variants were determined with sequencing, and global m6A levels were determined by using a competitive immunoassay method. We identified variants of concern (VOC; alpha, gamma, delta, omicron), the variant of interest (VOI; epsilon), and the lineage B.1.1.519. Global m6A methylation differed significantly across viral variants (p = 3.2 × 10-7). In particular, we found that m6A levels were significantly lower in the VOC delta- and omicron-positive individuals compared to non-infected individuals (p = 2.541236 × 10-2 and 1.134411 × 10-4, respectively). However, we uncovered no significant correlation between global m6A levels and viral nucleocapsid (N) gene expression or age. Furthermore, individuals with complete vaccination schemes showed significantly lower m6A levels than unvaccinated individuals (p = 2.6 × 10-4), and differences in methylation levels across variants in unvaccinated individuals were significant (p = 3.068 × 10-3). These preliminary results suggest that SARS-CoV-2 variants show differences in global m6A levels.

Epigenetic Transgenerational Modifications Induced by Xenobiotic Exposure in Zebrafish.

Zebrafish (Danio rerio) is a well-established vertebrate model in ecotoxicology research that responds to a wide range of xenobiotics such as pesticides, drugs, and endocrine-disrupting compounds. The epigenome can interact with the environment and transform internal and/or external signals into phenotypic responses through changes in gene transcription. Environmental exposures can also generate epigenetic variations in offspring even by indirect exposure. In this review, we address the advantages of using zebrafish as an experimental animal model to study transgenerational epigenetic processes upon exposure to xenobiotics. We focused mostly on DNA methylation, although studies on post-translational modifications of histones, and non-coding RNAs related to xenobiotic exposure in zebrafish are also discussed. A revision of the methods used to study epigenetic changes in zebrafish revealed the relevance and reproducibility for epigenetics-related research. PubMed and Google Scholar databases were consulted for original research articles published from 2013 to date, by using six keywords: zebrafish, epigenetics, exposure, parental, transgenerational, and F2. From 499 articles identified, 92 were considered, of which 14 were selected as included F2 and epigenetic mechanisms. Current knowledge regarding the effect of xenobiotics on DNA methylation, histone modifications, and changes in non-coding RNAs expressed in F2 is summarized, along with key experimental design considerations to characterize transgenerational effects.

Regulation of antral follicular growth by an interplay between gonadotropins and their receptors.

Knowledge of the growth and maturation of human antral follicles is based mainly on concepts and deductions from clinical observations and animal models. To date, new experimental approaches and in vitro data contributed to a deep comprehension of gonadotropin receptors' functioning and may provide new insights into the mechanisms regulating still unclear physiological events. Among these, the production of androgen in the absence of proper LH levels, the programming of follicular atresia and dominance are some of the most intriguing. Starting from evolutionary issues at the basis of the gonadotropin receptor signal specificity, we draw a new hypothesis explaining the molecular mechanisms of the antral follicular growth, based on the modulation of endocrine signals by receptor-receptor interactions. The "heteromer hypothesis" explains how opposite death and life signals are delivered by gonadotropin receptors and other membrane partners, mediating steroidogenesis, apoptotic events, and the maturation of the dominant follicle.

The Placenta as a Target of Epigenetic Alterations in Women with Gestational Diabetes Mellitus and Potential Implications for the Offspring.

Gestational diabetes mellitus (GDM) is a pregnancy complication first detected in the second or third trimester in women that did not show evident glucose intolerance or diabetes before gestation. In 2019, the International Diabetes Federation reported that 15.8% of live births were affected by hyperglycemia during pregnancy, of which 83.6% were due to gestational diabetes mellitus, 8.5% were due to diabetes first detected in pregnancy, and 7.9% were due to diabetes detected before pregnancy. GDM increases the susceptibility to developing chronic diseases for both the mother and the baby later in life. Under GDM conditions, the intrauterine environment becomes hyperglycemic, while also showing high concentrations of fatty acids and proinflammatory cytokines, producing morphological, structural, and molecular modifications in the placenta, affecting its function; these alterations may predispose the baby to disease in adult life. Molecular alterations include epigenetic mechanisms such as DNA and RNA methylation, chromatin remodeling, histone modifications, and expression of noncoding RNAs (ncRNAs). The placenta is a unique organ that originates only in pregnancy, and its main function is communication between the mother and the fetus, ensuring healthy development. Thus, this review provides up-to-date information regarding two of the best-documented (epigenetic) mechanisms (DNA methylation and miRNA expression) altered in the human placenta under GDM conditions, as well as potential implications for the offspring.

The Development and Evaluation of Brain and Heart Cell Lines from a Marine Fish for Use in Xenobiotic-Induced Cytotoxicity Testing.

Two cell lines derived from the brain and heart of a Pacific white snook specimen (Centropomus viridis) were developed and evaluated in terms of their responsiveness to glyphosate-induced cytotoxicity. The cells were grown in Leibovitz-15 (L-15) medium supplemented with 10% fetal bovine serum (FBS) and were passaged 36 times. Growth was tested at different concentrations of FBS (5, 10 and 20%) at 27°C. The cell lines were cryopreserved at different passages and were successfully thawed, with a survival rate greater than 80% without detectable contamination. At passage 36, the cells were used to assess the deleterious effects of glyphosate, and cell proliferation was measured by direct counting and with the MTT assay. Similar LC50 values were obtained with both methods. Although the principles behind these two assessment methods differ, our results show that both are suitable for evaluating glyphosate toxicity. In addition, heart- and brain-derived cells showed similar sensitivity, suggesting that the same mode of action might be responsible for the toxicity of glyphosate at the cellular level. The newly developed Pacific white snook brain and heart cell lines could be useful to investigate cellular and molecular mechanisms of toxicity, satisfying the need to reduce the use of animals in experiments. Glyphosate-related toxicological data obtained in the present study will allow us to continue investigating the effects of this herbicide directly on brain and heart fish cells since similar studies have only been carried out on either live organisms or on human cell lines such as neuroblastoma, which are immortalised by oncogenes or similar.

In silico identification of excretory/secretory proteins and drug targets in monogenean parasites.

The Excretory/Secretory (ES) proteins of parasites are involved in invasion and colonization of their hosts. In addition, since ES proteins circulate in the extracellular space, they can be more accessible to drugs than other proteins, which makes ES proteins optimal targets for the development of new and better pharmacological strategies. Monogeneans are a group of parasitic Platyhelminthes that includes some pathogenic species problematic for finfish aquaculture. In the present study, 8297 putative ES proteins from four monogenean species which genomic resources are publicly available were identified and functionally annotated by bioinformatic tools. Additionally, for comparative purposes, ES proteins in other parasitic and free-living platyhelminths were identified. Based on data from the monogenean Gyrodactylus salaris, 15 ES proteins are considered potential drug targets. One of them showed homology to 10 cathepsins with known 3D structure. A docking molecular analysis uncovered that the anthelmintic emodepside shows good affinity to these cathepsins suggesting that emodepside can be experimentally tested as a monogenean's cathepsin inhibitor.

Congenital Malformations in Sea Turtles: Puzzling Interplay between Genes and Environment.

The completion of embryonic development depends, in part, on the interplay between genetic factors and environmental conditions, and any alteration during development may affect embryonic genetic and epigenetic regulatory pathways leading to congenital malformations, which are mostly incompatible with life. Oviparous reptiles, such as sea turtles, that produce numerous eggs in a clutch that is buried on the beach provide an opportunity to study embryonic mortality associated with malformations that occur at different times during development, or that prevent the hatchling from emerging from the nest. In sea turtles, the presence of congenital malformations frequently leads to mortality. A few years ago, a detailed study was performed on external congenital malformations in three species of sea turtles from the Mexican Pacific and Caribbean coasts, the hawksbill turtle, Eretmochelys imbricata (n = 23,559 eggs), the green turtle, Chelonia mydas (n = 17,690 eggs), and the olive ridley, Lepidochelys olivacea (n = 20,257 eggs), finding 63 types of congenital malformations, of which 38 were new reports. Of the three species, the olive ridley showed a higher incidence of severe anomalies in the craniofacial region (49%), indicating alterations of early developmental pathways; however, several malformations were also observed in the body, including defects in the carapace (45%) and limbs (33%), as well as pigmentation disorders (20%), indicating that deviations occurred during the middle and later stages of development. Although intrinsic factors (i.e., genetic mutations or epigenetic modifications) are difficult to monitor in the field, some environmental factors (such as the incubation temperature, humidity, and probably the status of feeding areas) are, to some extent, less difficult to monitor and/or control. In this review, we describe the aetiology of different malformations observed in sea turtle embryos, and provide some actions that can reduce embryonic mortality.

Molecular Effects of Silver Nanoparticles on Monogenean Parasites: Lessons from Caenorhabditis elegans.

The mechanisms of action of silver nanoparticles (AgNPs) in monogenean parasites of the genus Cichlidogyrus were investigated through a microarray hybridization approach using genomic information from the nematode Caenorhabditis elegans. The effects of two concentrations of AgNPs were explored, low (6 µg/L Ag) and high (36 µg/L Ag). Microarray analysis revealed that both concentrations of AgNPs activated similar biological processes, although by different mechanisms. Expression profiles included genes involved in detoxification, neurotoxicity, modulation of cell signaling, reproduction, embryonic development, and tegument organization as the main biological processes dysregulated by AgNPs. Two important processes (DNA damage and cell death) were mostly activated in parasites exposed to the lower concentration of AgNPs. To our knowledge, this is the first study providing information on the sub-cellular and molecular effects of exposure to AgNPs in metazoan parasites of fish.

Effects of feed restriction on growth performance, lipid mobilization, and gene expression in rose spotted snapper (Lutjanus guttatus).

The effects of feed deprivation were evaluated for 1 week and 2 weeks in Lutjanus guttatus juveniles. A significant reduction in body mass was observed in both feed deprivation schemes, as well as in hepatosomatic, viscerosomatic and mesenteric fat indexes. The composition of fasted fish was characterized by a decrease in lipid content; the liver displayed an intense reduction of lipid reserves in both fasted groups, and increased expression of the lysosomal acid lipase. 1 week after re-feeding, both experimental groups showed an increase in specific growth rate, feed intake and feed conversion ratio. A recovery in hepatic lipid reserves was observed, and the expression of the lysosomal acid lipase decreased, although lipid content in both groups was still significantly lower than in control groups. Hepatic expression of the growth hormone receptor decreased after fasting, and remained low even after the fish were fed, whereas the expression of insulin-like growth factor 1 and 2 increased after fasting and decreased in both groups when fish were fed again. Altogether, these results showed a partial compensatory growth response in L. guttatus juveniles after fasting, with enhanced growth rates and improved feed efficiency. Fish used stored lipid reserves as the main energy source, and the expression of growth-related and lipid mobilization marker genes in the liver showed similar patterns in both fasting schemes. Based on the results, we suggest as much as 1-week fasting intervals during grow-out programmes to reduce visceral fat and increase growth rate in this species.

Post-transcriptional silencing of myostatin-1 in the spotted rose snapper (Lutjanus guttatus) promotes muscle hypertrophy.

Muscle growth is regulated by several factors including the growth differentiation factor 8, known as myostatin, an inhibitor of myocyte differentiation and proliferation. Research on myostatin regulation was already conducted to improve growth rates in farmed animals, including aquatic species. To explore the effects of myostatin inactivation in a commercial marine fish (spotted rose snapper, Lutjanus guttatus) in vivo, we induced post-transcriptional silencing (knockdown) of myostatin-1 (mstn-1) by injecting dsiRNA directly into the muscle of juvenile fish (87 days post-hatch) using a commercial polymer as vehicle. Results show a significant decrease in mstn-1 expression starting at 2 days after injection and for up to 5 days. Knockdown of mstn-1 caused muscle fiber hypertrophy (but not hyperplasia); however, there were no significant changes in weight or length. Although still experimental, this study provides evidence that temporary knockdown of mstn-1 in a commercial marine fish in vivo promotes fiber hypertrophy and therefore could potentially help grow-out programmes in fish aquaculture.

Genome-wide identification of ABC transporters in monogeneans.

ATP-Binding Cassette (ABC) transporters are proteins that actively mediate the transport of a wide variety of molecules, including drugs. Thus, in parasitology, ABC transporters have gained attention as potential targets for therapeutic drugs. Among the parasitic Platyhelminthes, ABC transporters have been identified and classified in a few species of Trematoda and Cestoda but not in Monogenea. Monogeneans are mainly ectoparasites of marine and freshwater fish, although they can also be found on other aquatic organisms. Severe epizootics caused by monogeneans have been reported around the world, mainly in confined and/or overcrowded fish. The purpose of this study was to identify the ABC transporters in four species of monogeneans (Gyrodactylus salaris, Protopolystoma xenopodis, Eudiplozoon nipponicum and Neobenedenia melleni) for which genomic resources are publicly available. For comparative purposes, ABC transporters were also identified in endoparasitic (Schistosoma mansoni and Echinococcus granulosus) and free-living (Macrostomun lignano and Schmidtea mediterranea) platyhelminths. Thirty-two putative ABC transporters were identified in the genome of G. salaris, 40 in the genome of P. xenopodis, 46 in the transcriptome of E. nipponicum and 9 in a rather limited ESTs set available for N. melleni. Of the eight ABC subfamilies (A-H) known in metazoans, subfamily H was the only one not found in any monogenean species. In contrast, ABCC was the best represented subfamily. Phylogenetic analyses showed a few cases of one-to-one orthologous relationships, which agree with results from other metazoan species. We found some monogenean ABC members related to subfamilies B, C and G involved in drug resistance in humans. This information may be useful for future functional studies on ABC transporters in monogeneans.

Mercury Concentration, DNA Methylation, and Mitochondrial DNA Damage in Olive Ridley Sea Turtle Embryos With Schistosomus Reflexus Syndrome.

Schistosomus reflexus syndrome (SR) is a rare and lethal congenital malformation that has been reported in the olive ridley sea turtle (Lepidochelys olivacea) in Mexico. Although the etiology remains unclear, it is presumed to be genetic. Since embryonic development in sea turtles largely depends on environmental conditions, we investigated whether sea turtle total mercury content participates in the etiology of SR. Given that several toxins are known to affect both DNA methylation and/or mitochondrial DNA (mtDNA) copy number, we also probed for associations of these parameters to SR and mercury exposure. We measured the levels of each variable in malformed olive ridley sea turtle embryos (either with SR or other non-SR malformations) and embryos without malformations. Malformed embryos (with or without SR) showed higher mercury concentrations compared to normal embryos, while only embryos with SR showed higher levels of methylation compared to embryos without malformations and those with other malformations. Furthermore, we uncovered a positive correlation between mercury concentrations and DNA methylation in SR embryos. With respect to mtDNA copy number, no differences were detected across experimental groups. Because of sample size limitations, this study is an initial attempt to understand the association of environmental toxins (such as mercury) and epigenetic alterations (DNA methylation) in the etiology of SR in sea turtles.

Overfeeding a High-Fat Diet Promotes Sex-Specific Alterations on the Gut Microbiota of the Zebrafish (Danio rerio).

Diet modulates the gut microbiota and is one of the main factors promoting obesity and overweight. In the present study, we investigated the effect of a high-fat diet (HFD) on the gut microbiota of the zebrafish (Danio rerio). Fish were separated into three groups and fed in different regimes: low fat, high fat, and high fat overfed; the experiments were performed on males and females separately. We analyzed more than 2.6 million sequences of variable region V3 of the 16S rRNA gene generated by the Illumina Miniseq platform, clustered to 97% similarity with vsearch and classified with the EzBioCloud database. The weight gain, condition factor (K), and body mass index were calculated as indicators of obesity. Multivariate analysis (PERMANOVA and ANOSIM) and diversity indices (Shannon and Dominance) revealed that overfeeding a HFD disturbs the gut microbiota differently in males and females suggesting that sex is a significant factor (p < 0.05) for the composition of the gut microbiota of zebrafish. The results also indicate that a HFD provided in a basal caloric regime does not promote obesity or alterations in the gut microbiota.

Multiple biomarker approach in the fiddler crab to assess anthropogenic pollution in tropical coastal environments.

CONTEXT: Fiddler crabs are important to the ecology of estuarine systems around the world, however, few studies have incorporated them as bioindicators. Urias estuary represents one of the most urbanized lagoons in the Gulf of California region and received discharges from different sources: shrimp farm, thermoelectric plant, fish processing plants, and untreated domestic and sewage wastes. OBJECTIVE: Assess the effects on anthropogenic contamination on female fiddler crabs reproduction, survival and genetic stability. METHODS: Exposition of wild crabs from a less impacted (reference) site to naturally contaminated sediments on under controlled laboratory conditions. Reproductive parameters, levels of DNA damage and mortality rates were measured, together with chemical analyses of sediments. RESULTS: The most contaminated sediments corresponded to the site where fish processing plants were located and the integrated biomarker response analysis revealed that the most adverse effects were produced by exposure to sediments from this site; these crabs showed higher mortality (67%) and poorer ovarian development than those crabs exposed to sediments from other sites. CONCLUSIONS: Female crabs under pollution stress are able to trade-off reproduction for survival, and surviving animals were able to restore genetic stability possibly by activating DNA repair mechanisms. Multiple biomarker approach discriminates different coastal contamination scenarios.

Hox Genes in Reptile Development, Epigenetic Regulation, and Teratogenesis.

Reptiles are ancestral organisms presenting a variety of shapes, from the elongated vertebral column of the snake to the turtle dorsalized ribs or retractile neck. Body plans are specified by a conserved group of homeobox-containing genes (Hox genes), which encode transcription factors important in cell fate and vertebral architecture along the anteroposterior axis during embryonic development; thus, dysregulation of these genes may cause congenital malformations, from mild-sublethal to embryonic-lethal. The genetic pool, maternal transfer, and environmental conditions during egg incubation affect development; environmental factors such as temperature, moisture, oxygen, and pollution may alter gene expression by epigenetic mechanisms. Thus, in this review, we present information regarding Hox genes and development in reptiles, including sex determination and teratogenesis. We also present some evidence of epigenetic regulation of Hox genes and the role of the environment in epigenetic modulation of gene expression. So far, the evidence suggests that the molecular instructions encoded by Hox genes to build a snake, a lizard, or a turtle represent the interplay between genome and epigenome after years of evolution, with occasional environmentally induced molecular mistakes leading to abnormal body shapes.