Parental transgenerational epigenetic inheritance related to dietary crude oil exposure in Danio rerio

Organismal survival and performance depend upon the capacity to cope with stressors based on within-lifetime experiences as well as transgenerationally inherited parental contributions (Donelson et al., 2012; Stein et al., 2018). Variation in the offspring population of the parent-to-offspring inherited information may result in phenotypic variation that can become subject to natural selection (Bautista and Burggren, 2019; Dey et al., 2016). When a parental population is exposed to challenging environments, its influence on their progeny may be beneficial. For example, the incidence of maladaptive phenotypes is lessened (Clark et al., 2014), or their survival is enhanced (Bautista and Burggren, 2019), when the offspring face environmental challenges that are similar to the parental exposure. In contrast, parental exposure may also result in offspring with maladaptive traits, such as the increase in the presence of developmental deformities that can last for generations (Burggren and Dubansky, 2018; Corrales et al., 2014).

Parent-to-offspring transgenerational inheritance may occur through genetic (Cavalli and Heard, 2019; Clark et al., 2014) and/or non-genetic mechanisms (Burggren, 2016; Jablonka, 2017; Knecht et al., 2017). Although the transmission of genetic and epigenetic marks occurs from both the maternal and paternal lines (Emborski and Mikheyev, 2019), it is generally assumed that the maternal line has more opportunities to contribute to the survival and fitness of their offspring through the mechanism of provisioning with nutritive resources (Dey et al., 2016; Herman and Sultan, 2016; Ho et al., 2014; Siddique et al., 2017). Thus, experimental designs have been primarily focused on understanding the influence of solely the maternal line in offspring survival and performance (Pick et al., 2016). For example, in Atlantic silverside, the fatty acid composition in the yolk provided by the mother is directly correlated with survival during exposure to a high CO₂ environment (Snyder et al., 2018).

The potential role of paternal inheritance has thus been overshadowed even though, similarly to the maternal line, it may result in modified phenotypes that are substrate for selection (Siddique et al., 2017). As evidence, F₁ and F₂ offspring derived from zebrafish males exposed to bisphenol A showed a dose-dependent increase in embryo mortality and a higher incidence of body malformations (Lombó et al., 2015). Moreover, the DNA methylome in zebrafish embryos was inherited solely from the spermatozoids and not from oocytes (Jiang et al., 2013), highlighting the importance of paternal inheritance for offspring development and performance. Thus, inheritance from the paternal line may be an important mechanism for transmitting epigenetic marks to the offspring. Although the maternal and paternal lines can both be directly involved in offspring survival and fitness, we currently have only a limited understanding of the separate contributions of each line of inheritance for offspring performance, allowing them to cope with persistent environmental stressors. In addition, how the molecular processes are potentially involved in the regulatory mechanisms of phenotypes and their inheritance through male and female lineages deserves more attention.

Epigenetic regulation and its phenotypic consequences happen through different molecular processes that include the modification of histones and/or DNA methylation (Burggren, 2016; Jablonka and Lamb, 2017; Walker and Burggren, 2020). DNA methylation and the post-translational modification in histone N-terminals are major modifications in the epigenome associated with repression or activation of genes crucial for embryogenesis, cell differentiation and organismal development (Jiang et al., 2013; Toni and Padilla, 2016). Indeed, both of these modifications are involved in the inheritance of reproductive defects (Yang et al., 2018) and organismal transgenerational response to environmental and anthropogenic stressors (Falisse et al., 2018; Nilsson et al., 2018). Furthermore, environmental or anthropogenic stressors that modify these processes will potentially induce broad ecological impacts for population maintenance in the wild (Bhandari et al., 2015; Ryu et al., 2018).

Differentiating maternal from paternal effects takes on considerable significance given the burgeoning interest in understanding the transgenerational effects in fish of environmental stressors such as crude oil (Jasperse et al., 2019; Simning et al., 2019). Exposure to crude oil in fish may happen through their skin, gills and diet (Tierney et al., 2013). Crude oil is a persistent environmental stressor that affects fish at all developmental stages. Oil exposure affects development by inducing edema formation and body skeletal curvatures, compromising cardiac performance by reducing stroke volume, cardiac output and heart contractility, impairing swimming performance by reducing critical swimming speed, and altering reproductive behaviors, among other effects (Bautista et al., 2019; Burggren and Dubansky, 2018; Incardona and Scholz, 2018; Johansen et al., 2017; Mager et al., 2018; Pasparakis et al., 2019). Fish are generally able to detect the presence of oil in the water, and may be able to escape if the occurrence of oil is localized (Bøhle, 1986; Schlenker et al., 2019). Even if fish can escape, oil exposure can still occur through ingestion of contaminated prey (Olsvik et al., 2011), but without reducing their appetite (Bautista et al., 2019; Christiansen and George, 1995). Notably, dietary exposure to crude oil induces similar effects to waterborne exposures, highlighting its relevance as an environmental stressor (Corrales et al., 2014; Lucas et al., 2016; Nahrgang et al., 2019; Vieweg et al., 2018; Vignet et al., 2014).

Experiments investigating transgenerational implications of parental exposure to oil through experimentation with wild or wild-captured fish populations typically requires a long period of time and a large amount of resources and space, which is rarely practical. Consequently, little is currently known about the relative contribution of each parental line to their offspring's survival and performance. Therefore, to test the effects of parental acute and chronic exposures to environmental stressors on their offspring, the use of animal models has increased in several disciplines such as genetics, physiology, behavior and ecotoxicology (Burggren and Dubansky, 2018; Pitt et al., 2018; Zhou et al., 2019).

In this study, we used zebrafish [Danio rerio (Hamilton 1822)] as a model for a multilevel approach to disentangle maternal and paternal effects of dietary exposure to crude oil on offspring survival and performance. To test whether the parental population per se was affected by crude oil exposure, we measured phenotypic traits directly related to fish health (growth and heart rate). We also investigated whether oil exposure leads to epigenetic modifications of the heart and the gonads by quantifying the level of histone-related antibodies, global DNA methylation, and change in the regulation of genes involved in heart function and DNA methylation transfer.

Finally, early developmental stages are considered to be highly sensitive to environmental stressors (Mager et al., 2017; Reddam et al., 2017). Thus, zebrafish embryos and larvae represent a tractable model to assess organismal performance and the influence of parental inheritance during exposure to environmental stressors. In this context, we determined the influence of the maternal and paternal oil exposure on the phenotype of the offspring by measuring fitness-related traits (early survival, heart rate and hypoxia tolerance). Concurrently, in those same larval groups, analyses were made of histone modification, global DNA methylation, and change in the regulation of genes involved in heart development and function or DNA methylation maintenance.

Eighty adult (6 months old) AB strain zebrafish (40 females, 40 males), were obtained from a local supplier and maintained individually in 1 liter tanks at the University of North Texas. Before experimentation, the fish were divided into four groups of 20 fish each as follows: (1) non-oil-exposed females, (2) non-oil-exposed males, (3) oil-exposed females (receiving an oil-spiked diet) and (4) oil-exposed males (Fig. 1). Prior to exposure, the fish were acclimated for 2 weeks following recommended husbandry conditions for this species (∼27±0.5°C, pH ∼7.6, 14 h:10 h light:dark cycle, ∼7.8 DO mg l⁻¹) (Spence et al., 2008; Westerfield, 2007). The fish were fed 3% of body mass twice per day with commercial flake food (TetraMin Tropical food). All experiments described in this study were performed at the University of North Texas under Institutional Animal Care and Use Committee Protocol no. 15003.

To prepare the oiled diet used in this experiment, high energy water accommodated fractions (HEWAFs) of crude oil were prepared following standard protocols (Bautista et al., 2019; Forth et al., 2017; Incardona et al., 2013; Mager et al., 2014; Reddam et al., 2017). Briefly, 2000 mg of Source Oil B acquired from the Deepwater Horizon oil spill was added into 1 liter of conditioned aquarium water (60 mg l⁻¹ of Instant Ocean salts buffered at pH 7.6) and blended for 30 s in a commercial blender (Waring^TM CB15). The mixture was allowed to settle in a separation funnel for 1 h, after which 100 ml was drawn out through the bottom port of the funnel and discarded. A volume of 600 ml of the remaining solution was considered to be 100% HEWAF and was used to prepare the oiled diet used during this experiment.

Commercial flake food (2 g) was evenly spread across the bottom of plastic weighing boats (135×135×20 mm, length×width×height) and sprayed with either 5 ml of conditioned water (control food) or 5 ml of 100% HEWAF (oiled food). The spraying process was performed under a fume hood, where the food was allowed to dry for a subsequent 12 h. After drying, the food was collected and stored at 4°C in amber glass containers covered with aluminum foil. Concentrations of polyaromatic hydrocarbons (PAHs) of these diets have previously reported as 0 and 24.2 mg kg⁻¹ for the control and 100% HEWAF diets, respectively (Bautista and Burggren, 2019).

Female and male groups were fed twice daily (3% of body mass per feeding event) with their respective dietary treatment for 21 days (Fig. 1A). To avoid additional oil exposure through coprophagia or exposure via the gills, fish were allowed to eat for 10 min in each feeding event, after which the non-eaten food and feces were siphoned out and 300 ml of water was renewed.

Immediately after the oil exposure period, the parental groups were divided and fish were selectively mated to produce the following groups of offspring crosses: (1) control offspring, derived from non-exposed parents (C♀C♂); (2) paternally exposed offspring, derived from mating pairs where only males were exposed (C♀E♂); (3) maternally exposed offspring, derived from mating pairs where only the females were exposed (E♀C♂); and (4) dual-parental-exposed offspring, derived from mating pairs where both parents were exposed (E♀E♂) (Fig. 1B,C). Breeding was carried out by placing three females and three males from the indicated parental populations into 3 liter tanks. The fish were maintained separated by sex overnight, and the following morning, at the start of daylight (∼07:00 h), both sexes were placed together for 2 h for courtship and mating. After this period, the eggs were collected from each tank and rinsed with clean aquarium water at 27±0.5°C. Eggs obtained from the same type of parental crosses were mixed and inspected under stereoscopic microscopy to confirm fertilization and cell division of the embryos. Any non-viable embryos were discarded.

To test whether the parental oil exposure influenced offspring survival or heart rate, 200 eggs per parental cross (C♀C♂, C♀E♂, E♀C♂ or E♀E♂) were sampled and divided into eight subgroups of 25 individuals. Each subgroup was placed into a 40 ml beaker and allowed to develop to 5 days post-fertilization (dpf) in either clean water or 100% HEWAF (Fig. 1D,E). From the eight beakers derived from each parental cross, four beakers were exposed to each of the experimental conditions. Two of the beakers were used to estimate survival and the other two beakers were used to estimate heart rate (Fig. 1).

The remaining F₁ offspring from each parental cross were raised to 13 or 30 dpf in control clean water conditions for further physiological or molecular analysis (Fig. 1F,G). During this period, the larvae were fed twice a day with Otohime™ A1 and A2 commercial food (www.otohime.us). To maintain water quality, larvae were allowed to eat for 30 min during each feeding event, and the remaining food was then removed. All larval exposures and maintenance in this experiment were performed at 28±0.5°C.

Zebrafish larvae (13 dpf) raised under clean water from each of the different parental background groups were anesthetized with MS222 (100 mg l⁻¹) and 10 pools of 15 larvae per parental cross were sampled. The pools were directly frozen in liquid nitrogen and stored at −80°C until molecular analysis. Four to six 30 dpf larvae per condition were fixed in Z-fix solution (Anatech Ltd) for 24 h, rinsed and stored in PBS for 5 days before being transferred to 70% ethanol until histology analysis.

Fish standard body length was recorded at the end of the 2-week acclimation period and at the first, second and third week of oil exposure. To estimate body length, individual fish were photographed (Nikon Coolpix AW130, 16 Mpixels) and each picture was digitally analyzed with ImageJ software (https://imagej.nih.gov/ij/).

Loss of equilibrium (LOE) in larval and adult fishes is used as an indicator of resistance/susceptibility to stressors in fish (Galleher et al., 2010; Giacomin et al., 2019; Ho and Burggren, 2012; Puglis et al., 2019) and can be considered equivalent to ‘ecological death’, the point at which behavior is so altered that the fish cannot perform in any ecological context (Beitinger et al., 2000; Scott and Sloman, 2004). After breeding, individual adults from the various groups were placed individually in separated chambers (9×8×7 cm) contained in an aerated, temperature-controlled 120 liter water tank. The morning after an overnight acclimation period, all chambers were covered with a grid (to prevent fish from using aerial surface respiration) and transferred into a 40 liter tank of hypoxic water (10% O₂). The oxygen level of the water, which was controlled by bubbling nitrogen into the tank, was monitored with an oxygen meter (ProODO Optical Dissolved Oxygen Instrument, YSI, USA). Time to LOE for each fish was recorded starting at the moment the chambers were fully submerged in the hypoxic water. The time was recorded when the fish was unable to recover normal positioning after first losing its equilibrium. The fish were then quickly removed from the challenge tank, identified and transferred into a fully aerated tank for recovery.

One day after the recovery from the hypoxia challenge, adult fish were anesthetized with MS222 (300 mg l⁻¹), and their body mass and standard length were estimated. Three-quarters of each fish population was dissected. Each fish's ventricle and gonads were excised, and their wet mass was recorded. The ventriculo- and gonado-somatic indices were then calculated as a percentage of body mass. After mass determination, the tissue samples were immediately frozen in liquid nitrogen and stored at −80°C until processed for molecular analysis (see below). The individuals from the remaining quarter of each population were fixed with Z-fix aqueous solution (Anatech Ltd) for 24 h and then stored in phosphate-buffered saline (PBS) for 5 days. The liver, gut, ventricle and gonads were then excised. Wet mass of the organs was recorded and expressed as percentage of body mass to determine their specific index. Tissues were then stored in 70% ethanol until histological processing.

During the third week of exposure, heart rate was measured in the parental populations using an electrical impedance technique. Briefly, the fish (n=20 per condition) were lightly anesthetized in a solution of 100 mg l⁻¹ MS222. Fish lost their equilibrium within 1 min of being submerged, and at that moment we carefully removed them from the solution, weighed them, and individually placed them with the ventral side facing up in a sponge containing a V-shaped trench to receive the fish's body. This process took <30 s. To maintain the anesthesia state in the fish during the protocol, aerated and temperature-controlled (28±0.5°C) fresh conditioned water (60 mg l⁻¹ Instant Ocean salts buffered at pH 7.8 with HCO₃⁻) containing 90 mg l⁻¹ of MS222 was delivered into the fish's mouth at a rate of 28±2 ml min⁻¹. Delivery occurred via a tubing tipped by a blunt 24 G gauge needle placed in the mouth to direct a flow of water across the gills. Two monopolar needle electrodes (Ecvel-Tech, Ltd) were carefully positioned on the ventral skin over the cardiac area of the fish. The electrodes were then connected to an impedance converter (model 2991, UFI, Morro Bay, CA, USA), connected to an amplifier (model PL3504, PowerLab 4/35) and the signals were recorded using LabChart software V7. The entire apparatus was replicated to allow measurements in four fish at a time.

Anesthetized adult fish placed in the heart rate measurement apparatus were allowed to stabilize for 30 min prior to intraperitoneal injections (Sidhu et al., 2014). We attempted to trigger maximum heart rate in the fish by first injecting atropine sulfate (1.2 µg g⁻¹ of fish) to increase heart rate by blocking the activity of cholinergic fibers, thus stopping the ‘parasympathetic drive’ that would depress heart rate. Fifteen minutes after this injection, isoproterenol, a non-selective β-adrenergic agonist, was injected at a dose of 7.8 ng g⁻¹ body mass to maximize the sympathetic influence of the nervous system on cardiac contractility and trigger the maximum heart rate frequency. After another 15 min, an injection of the non-selective β-adrenergic antagonist propranolol (1.2 µg g⁻¹ of fish) was made to decrease heart rate and contractility as a result of to competition with isoproterenol. All injected drugs were dissolved in physiological saline solution (0.9% NaCl) and delivered through the same body location. The three injections collectively accounted for ∼3% of fish body mass. Heart rate signals were recorded for each fish at 1 min intervals for 10 time points during the experiment: at 5 min before the atropine injection, and at 5, 10 and 15 min after each of the three drug injections. At the end of the procedure, the fish were carefully recovered and returned to their rearing tanks containing well-aerated conditioned water. A 75% survival rate was observed at the end of these experiments.

Survival of the F₁ offspring derived from each parental cross was estimated for 5 days during exposure to clean water or 100% HEWAF. Survival was assessed daily from fertilization through 5 dpf. Larvae were considered dead when absence of heartbeat was observed under stereoscopic microscopy.

At 12 and 30 dpf, 20 zebrafish larvae from the different parental backgrounds, all raised in clean water, were placed individually in small cylindrical glass chambers (4.0×0.6 cm) partially sealed at both ends with fine netting that kept the fish within the tube but allowed water to freely move through the tube. Groups of four chambers were then transferred into a 10 liter tank containing hypoxic water (13% O₂). The oxygen level of the water was controlled by nitrogen bubbling into the tank and monitored with an oxygen meter (ProODO Optical Dissolved Oxygen Instrument, YSI). The time to LOE for each larvae was recorded starting at the moment when the chambers fully entered into the hypoxic water and ending when the larvae had lost equilibrium for three consecutive seconds without recovery. Larvae were then quickly removed from the hypoxic water and transferred into a tank containing fully aerated water for recovery.

Heart rate (beats min⁻¹) of the offspring population was measured on a daily basis under stereoscopic microscopy (Nikon SMZ1000) during the 5 days of exposure. Temperature (28±0.5°C) and oxygen levels (7.5±0.2 mg l⁻¹) during the measurement period were determined with an oxygen meter (ProODO Optical Dissolved Oxygen Instrument, YSI). Video recordings (20 s) were acquired of larvae from each parental group and exposure condition within the first 4 h from the start of the photoperiod (07:00–11:00 h) every morning. The videos were subsequently processed with Photoshop CS6 extended version and analyzed using ImageJ software.

Zebrafish larvae (13 dpf) raised under clean water from each of the different parental background groups were anesthetized with MS222 (100 mg l⁻¹) and 10 pools of 15 larvae per parental cross were sampled. Each larvae pool was directly frozen in liquid nitrogen and stored at −80°C until molecular analysis. Four to six 30 dpf larvae per condition were fixed in Z-fix solution for 24 h, rinsed and stored in PBS for 5 days and then transferred to 70% ethanol until histology analysis.

Heart and gonadal tissues from female and male adults and 30 dpf larvae were stored in 70% ethanol prior to processing for histological analysis following standard procedures (Carson, 1990). In brief, the tissues were dehydrated with a series of ethanol baths after which they were washed three times in Histochoice^® clearing agent. After clearing, the tissues were embedded in paraffin and subsequently sliced with a manual microtome (Leica RM2245) in 5 µm sections.

To determine whether exposure to crude oil leads to changes in the levels of histones associated with gene expression or silencing in both populations, hearts and gonads from three males and females of each parental group, as well as from three 30 dpf larvae from each parental cross, were immunohistologically stained. Antibodies were revealed using the VECTASTAIN^® Elite^® ABC Universal Plus kit, Peroxidase (Horse Anti-Mouse/Rabbit IgG; PK-8200) following the manufacturer's instructions. Essentially, after preparing the slides of these organs and larvae, sections were deparaffinized and hydrated through a series of Histochoice^® and graded alcohol series. After unmasking and quenching, the sections were incubated with mouse or rabbit primary antibodies: rabbit polyclonal (mAbcam 8580) anti-histone H3 (tri methyl K4, epigenetic marker for promoters of active genes and initiated forms of RNA polymerase II; H3K4me3), and mouse monoclonal (mAbcam 6002) anti-histone H3 (tri methyl K27, epigenetic marker for inactive genes; H3K27me3) (Toni and Padilla, 2016). After staining, the samples were imaged with light microscopy (Zeiss Axio Imager.M2) fitted with an AxioCam camera MRc.

Indirect determination of tissue antibody levels was performed by digital analysis (ImageJ). All images were acquired using the same microscopy parameters (scale, zoom, opening of diaphragm, lighting). Control reagent stains were performed for each antibody and each slide to determine the level of background staining in a 5000 µm² area. Based on the control reagent stains, the color threshold values for positive signals were determined for each antibody at 40× (HUE ratio 0:255; saturation 0:255; and brightness ratio 40:113) and used to standardize the analysis. After setting these parameters for each image, the function ‘Analyze Particles’ in ImageJ was used to measure the relative color intensity in the samples.

Assessment of global DNA methylation of cardiac and gonadal tissues of the parental population was performed in pools of three individuals from the same group and sex of each parental population. For DNA and RNA extractions in the parental population, three hearts (or three gonads) were homogenized using mortar and pestle following a protocol reported elsewhere (Tate et al., 2016). Each pool was then split into two equal parts, and DNA and total RNA were extracted using DNAzol and RNAzol (Molecular Research Center) solutions according to the manufacturer's protocols. The integrity of the extraction was assessed by electrophoresis on 1% agarose gel. DNA and total RNA of the F₁ offspring population were extracted with a similar protocol to the parental population, but nine pools (15 larvae each) per parental cross were used.

Purity and concentrations of DNA and RNA were assessed using a Nanodrop 2000 Spectrophotometer (Nanodrop, Thermo Fisher Scientific, USA). All samples were stored at −80°C until further processing.

Relative levels of global DNA methylation (5-methylcytosine) percentage in the DNA samples were quantified using the MethylFlash™ Methylated DNA Quantification Kit (Epigentek Group, USA), according to the manufacturer's instructions. OD intensity of each DNA sample was normalized to 100 ng. This protocol was used for both adult and larval groups.

Total RNA obtained from the different samples (pools of three hearts or three gonads, for the parental population, and pools of 15 larvae in the F₁ offspring) was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) and kept at −20°C until use. Quantitative real-time polymerase chain reaction (qPCR) was used to analyze one candidate gene involved in the response to PAHs (CYP1a), four genes involved in cardiac development (GATA4, MyH7, MyL7 and NKX2.5) and three genes involved in cardiac stress (NPPA, TNNT2a and ATP2a2a). Additionally, 10 genes were analyzed that are specifically involved in DNA methylation, specifically, maintenance (DNMT1), establishment of new methylation (DNMT3, DNMT4, DNMT5, DNMT6 and DNMT7), methylation transfer (GNMT), and demethylation (TET1, TET2 and TET3). Primer design for each gene was performed using Primer3Plus software based on cDNA sequences available in the public database NCBI (https://www.ncbi.nlm.nih.gov) (Table S1). qPCR was performed using Evagreen Supermix (Bio-Rad Laboratories, USA). The genes WDTC1 and ACTB2 were chosen as reference (‘housekeeping’) genes for the gonads and heart, respectively, because they showed minimal variability in their transcript levels, both within and among treatments. ACTB2 was also used as the housekeeping gene for the offspring. No amplification was observed in negative controls. The CFX Manager program (Bio-Rad Laboratories, USA) was used to determine the threshold cycle (C_t) values. The qPCR efficiency (E) was calculated for each gene according to the equation E=10^(−1/slope). The quantification of the transcript was then calculated using a correction of efficiency for each transcript and indicated relative to the housekeeping gene expression (Pfaffl, 2001).

All data were tested for normality and homogeneity of variances using Kolmogorov–Smirnov and Brown–Forsythe tests, respectively. If the data did not meet these assumptions, non-parametric analyses were performed and reported (Quinn and Keough, 2002).

In the parental generation, body length was compared among parental groups with a three-way ANOVA, using oil exposure, sex and time as factors. Time to LOE under hypoxia was analyzed using two-way ANCOVA with diet exposure and sex as factors and body mass as a covariate. Heart rate was analyzed using three-way ANCOVA using oil exposure, sex and time as factors, and mass as covariate. The hepato-somatic, gut-somatic, ventriculo-somatic and gonado-somatic indices were analyzed using two-way ANOVAs with diet and sex as factors. Antibody quantity, global DNA methylation level and gene expression profiles in the heart and gonads were analyzed using two-way ANOVAs with diet and sex as factors.

Larval survival was analyzed with the Cox stratified model using the F₁ exposure condition as strata and maternal and paternal exposures as covariates. Because differences were found among groups, survival log-rank tests were employed to analyze each exposure condition by separate and the Holm–Šidák method was subsequently employed for multiple comparisons. Heart rates in larvae were rank transformed and analyzed with a general linear model (GLM) – multifactor ANOVA, using time, oil exposure, and maternal and paternal exposure experience as categorical factors. Because the interaction of the factors induced significant differences (P=0.011), we subsequently analyzed each F₁ exposure condition (clean water and 100% HEWAF) separate using a GLM – three-way ANOVA, where time and parental exposure conditions were employed as factors. Time to LOE under hypoxia was rank transformed for the larvae, and a three-way ANOVA was performed on the ranks, with maternal exposure, paternal exposure and developmental age as factors. Antibody quantity at 30 dpf, global DNA methylation level at 13 dpf and gene expression profiles were analyzed using two-way ANOVAs with maternal exposure and paternal exposure as factors.

The a posteriori Tukey's honest significant difference (HSD) test was used for mean comparisons. Statistica version 7.0 (StatSoft, USA), Sigmaplot V.14 and Statgraphics centurion XVI software were used to analyze the data. Statistical significance was considered at an α-value of 0.05 for all statistical tests. All data are expressed as means±s.e.m. unless otherwise indicated.

No mortality owing to dietary oil exposure was observed in the parental population. Standard body length varied from 28.9±0.4 and 28.4±0.4 mm for female and male fish, respectively, at the end of the initial acclimation period, increasing slightly to 31.6±0.5 and 31.2±0.5 mm, respectively, at the end of the oil exposure period. There were no significant differences in length (P>0.69) or mass (P=0.46) between experimental groups. Average adult body mass was 0.46±0.01 g at the end of the experiment.

Diet and sex did not significantly affect time to LOE under hypoxia in the parental generation (diet, F_1,88=0.003, P=0.95; sex, F_1,88=0.009, P=0.92). The average time to LOE of all adults was 120±6 min (Fig. 2A).

Diet and sex had a significant effect on heart rate frequency in the parental generation (diet, F_1,689=0.05, P=0.82; sex, F_1,689=7.30, P=0.007), but no differences were revealed in post hoc tests (Fig. 6A). However, there was a significant effect (F_9,689=5.14, P<0.001) of the time points at which heart rate was measured. Also, heart rate measured under isoproterenol (producing maximum heart rate) was significantly higher than the heart rate measured under atropine and after the propranolol injection (Table S2).

The hepato-somatic index of adults was not significantly affected by diet (F_1,22=0.22, P=0.64) or sex (F_1,22=3.19, P=0.09) (Table 1). Similar results were observed for the gut-somatic index, in which no difference was observed according to diet (F_1,24=0.13, P=0.73) or sex (F_1,24=0.02, P=0.89). However, sex had a significant effect on the ventriculo-somatic index (F_1,122=9.60, P=0.002) and the gonado-somatic index (F_1,111=226.57, P<0.001), with females having a smaller ventriculo-somatic index but higher gonado-somatic index compared with males (Table 1). Oil exposure via diet had no effect on the ventriculo-somatic index (F_1,122=0.03, P=0.85) or the gonado-somatic index (F_1,111=0.04, P=0.85).

There was a significant effect of the interaction between dietary oil exposure and sex on the relative color intensity of H3K4me3, associated with gene activation in heart tissue (F_1,8=351.5, P<0.001; Fig. 3A). The relative color intensity of H3K4me3 was reduced in the exposed fish compared with control fish, but within the control groups, the male fish exhibited increased relative color intensity of H3K4me3 compared with the female fish. However, the color intensity of H3K27me3, associated with gene silencing, was significantly increased in the hearts of exposed fish (F_1.8=578.5, P<0.001; Fig. 3B). In addition, males exhibited higher relative color intensity of H3K27me3 in each diet exposure (F_1,8=161.3, P<0.001).

In gonadal tissue, we observed a significant effect of the interaction between diet and sex on color intensity of both histone-related antibodies (F_1,8=75.9, P<0.001 and F_1,8=7.1, P=0.28, for H3K4me3 and H3K27me3, respectively; Fig. 3C,D). Control and exposed females were similar, whereas antibody relative color intensity was higher in control males and significantly even higher in exposed males.

Dietary oil exposure significantly reduced cardiac global DNA methylation in the parental generation (F_1,20=5.00, P=0.04). Oil-exposed adults exhibited a lower percentage of global DNA methylation than control fish (Fig. 4A,B). In contrast, sex did not significantly impact cardiac global DNA methylation levels (F_1,20=2.06, P=0.17). Additionally, there was no significant effect on gonadal global DNA methylation levels of either oil exposure (F_1,20=0.07, P=0.79) or sex (F_1,20=0.61, P=0.44) (Fig. 4B).

No significant effects of oil exposure in adults were observed in cardiac transcripts levels of genes involved in oil response (CYP1a, F_1,15=0.14, P=0.71), cardiac development (GATA4, F_1,16=0.11, P=0.75; MyH7, F_1,16=1.81, P=0.20; MyL7, F_1,16=0.74, P=0.40; NKX2.5, F_1,16=0.11, P=0.75) or cardiac stress (NPPA, F_1,15=0.0002, P=0.99; TNNT2a, F_1,14=0.60, P=0.45; ATP2a2a, F_1,16=0.20, P=0.66). However, sex significantly impacted cardiac transcript levels of CYP1a (F_1,15=4.99, P=0.04) and ATP2a2a (F_1,16=1.06, P=0.02), in which males exhibited higher levels of transcript than females (Table 2).

In gonads, only the expression level of MyL7 (cardiac development) was significantly downregulated in oil-exposed fish compared with controls (F_1,14=4.94, P=0.04) (Table 2). There was no significant difference of oil exposure on gonadal transcript levels of all other genes (GATA4, F_1,16=0.01, P=0.91; MyH7, F_1,12=0.36, P=0.56; NKX2.5, F_1,16=1.48, P=0.24; NPPA, F_1,14=1.18, P=0.30; TNNT2a, F_1,15=1.00, P=0.33; ATP2a2a, F_1,16=0.59, P=0.45), including the one involved in the response to PAHs (CYP1a, F_1,16=1.93, P=0.18). Transcript levels of ATP2a2a were upregulated in females compared with males (F_1,16=10.79, P=0.005). In contrast, transcript levels in males were upregulated compared with females for MyH7 (F_1,12=18.21, P=0.001), MyL7 (F_1,14=19.67, P=0.001), NKX2.5 (F_1,16=12.49, P=0.003) and NPPA (F_1,14=52.17, P<0.0001).

Expression of GNMT (involved in the transfer of DNA methylation) was significantly upregulated in hearts of adult oil-exposed fish compared with hearts of control fish (F_1,16=8.05, P=0.01) (Table 3). In contrast, expression levels of TET3 (involved in DNA demethylation) were downregulated in hearts of exposed fish compared with controls (F_1,16=4.78, P=0.04). However, oil exposure in adults did not significantly alter cardiac expression of other measured genes: DNMT1 (F_1,16=2.34, P=0.15), the de novo methylation DNMT3 (F_1,15=2.12, P=0.17), DNMT4 (F_1,16=1.84, P=0.19), DNMT5 (F_1,16=1.66, P=0.22), DNMT6 (F_1,15=0.11, P=0.75), DNMT7 (F_1,16=1.81, P=0.20), TET1 (F_1,16=0.24, P=0.63) or TET2 (F_1,16=0.25, P=0.62). Nonetheless, the hearts of the females exhibited higher expression level of DNMT5 than those of males (F_1,16=12.23, P=0.003).

In adult gonadal tissue, DNMT1 was significantly influenced by the interaction between oil exposure and sex (F_1,16=5.24, P=0.04). Oil-exposed females had a higher transcript level than control females and both control and exposed males (Table 3). Transcript levels of all other genes were not significantly affected by diet (DNMT3, F_1,16=3.90, P=0.07; DNMT4, F_1,15=0.25, P=0.62; DNMT5, F_1,15=0.63, P=0.44; DNMT6, F_1,15=0.87, P=0.36; DNMT7, F_1,16=1.54, P=0.23; GNMT, F_1,16=0.02, P=0.89; TET1, F_1,16=1.38, P=0.25; TET2, F_1,16=1.70, P=0.21; TET3, F_1,16=0.07, P=0.79). In contrast, sex of the fish was associated with a significant effect on DNMT3 (F_1,16=6.06, P=0.03), with levels being higher in females compared with males. Similarly, transcript levels of males were higher than for females for DNMT6 (F_1,15=21.88, P=0.0003), TET1 (F_1,16=27.39, P<0.0001), TET2 (F_1,16=33.50, P<0.0001) and TET3 (F_1,16=11.91, P=0.003).

Survival rates of larval populations were differentially influenced by both parental oil exposure and offspring experimental conditions (P=0.039) (Fig. 5). During offspring exposure to clean water, survival of larvae derived from the control (C♀C♂) parental condition decreased from 100% to 94% from the first to the second day of exposure and stayed at this value throughout the experimental period (Fig. 5A). Survival rates from paternally exposed (C♀E♂) and dual-parent-exposed (E♀E♂) offspring groups were significantly different (P<0.001) from controls (C♀C♂) and decreased from 100% to 92% and 100% to 84%, respectively, at the end of the exposure. In contrast, the maternally exposed (E♀C♂) offspring group did not differ from controls (C♀C♂) and maintained a survival rate of 94% throughout the measuring period.

Oil exposure of offspring to crude oil via their ambient water induced a prominent decrease in survivorship in controls (C♀C♂) from 100% to 44% from the first to the third day of exposure and reaching 0% at the fourth day (Fig. 5B.). Notably, the offspring derived from the maternally (E♀C♂), paternally (C♀E♂) or dual-parent-exposed (E♀E♂) larval groups exhibited enhanced survival rates when compared with the control (C♀C♂) group, in which all individuals died by day 4 day of oil exposure (Fig. 5B). Remarkably, offspring derived from the maternally exposed group exhibited high survival values (∼80%) in the last 3 days of exposure (Fig. 5B).

Time to LOE produced by hypoxia was significantly affected by larval age (F_1,113=58.62, P<0.001). In 12 dpf larvae, time to LOE was just 5.4±0.4 min compared with 16.2±1.6 min in 30 dpf larvae (Fig. 2B). No significant difference occurred between parental oil treatments (F_3,113=1.81, P=0.15).

When maintained in clean water, offspring derived from the maternally oil-treated group (E♀C♂) exhibited bradycardia as early as 1 dpf compared with the other three groups (P=0.0001) (Fig. 6B), but this difference disappeared at 2 dpf. Also, at 2 dpf, paternally treated offspring (C♀E♂) exhibited higher heart rates compared with the dual-parent-exposed (E♀E♂) group when in clean water. In addition, at 5 dpf, offspring obtained from all oil-treated parental groups exhibited decreased heart rate in comparison with offspring obtained from the control parental group (C♀C♂) (Fig. 6B).

In comparison with offspring raised in clean water, exposure to 100% HEWAF induced bradycardia in all of the offspring groups (P=0.04). Although there was a time effect in which the four groups exhibited higher heart rates at 2 dpf compared with the rest of the exposure days (P=0.005), no difference was observed among the groups within any day of oil exposure (P>0.05) (Fig. 6C).

Maternal and paternal exposure conditions significantly affected H3K4me3 antibody relative color intensity of larval cardiac tissue (F_1,8=257.14, P<0.001). Color intensity of cardiac H3K4me3 was higher in the groups derived from the parental crosses in which one or both parents were exposed to crude oil (Fig. 7A). Nonetheless, relative color intensity of H3K4me3 antibody among offspring derived from crosses where only the mother or father was oil-exposed were not significantly different. Levels of H3K27me3 in larval hearts were not significantly different among groups (F_1,8=1.2, P=0.30; Fig. 7B).

Maternal and paternal exposure condition had significant effects on both H3K4me3 and H3K27me3 relative color intensity in larval muscle tissue (F_1,8=15.9, P=0.004, and F_1,8=18.869, P=0.002, respectively). H3K4me3 color intensity was higher in the maternally exposed (E♀C♂) and dual-parent-exposed (E♀E♂) larval groups compared with controls (C♀C♂) or the paternally exposed (C♀E♂) offspring groups (Fig. 7C). H3K27me3 relative color intensity was significantly higher in the paternally exposed (C♀E♂) group compared with the rest of the offspring (Fig. 7D).

The interaction between the maternal and paternal exposure significantly affected the global DNA methylation level of 13 dpf larvae (F_1,29=6.20, P=0.02). Larvae derived from maternally, paternally or dual-parent treated groups exhibited similar lower percentage of global DNA methylation compared with F₁ larvae derived from control parents (Fig. 4B).

The interaction between maternal and paternal oil exposure significantly altered larval F₁ expression of GATA4 and NPPA (F_1,20=9.51, P=0.006; F_1,20=10.65, P=0.004; respectively). The transcript levels of the genes of the 13 dpf larvae derived from the paternally treated (C♀E♂) group were upregulated compared with those in larvae derived from the control parents (C♀C♂) or from the dual-parent oil-treated group (E♀E♂) (Table 2). Additionally, maternal oil exposure affected NKX2.5 transcript levels (F_1,20=4.67, P=0.04). Larvae derived from the maternally oil-treated group showed reduced transcript levels compared with larvae derived from control mothers. The transcripts levels of all the other genes (MyH7, F_1,20=3.26, P=0.09; MyL7, F_1,19=0.91, P=0.35; TNNT2a, F_1,14=0.65, P=0.43; ATP2a2a, F_1,20=0.98, P=0.34), including the one involved in the response to PAHs (CYP1a, F_1,19=0.51, P=0.48), were not significantly affected by parental exposure background.

Finally, parental oil exposure did not significantly affect expression of any genes involved in methylation modification in the offspring generation (DNMT1, F_1,19=0.16, P=0.69; DNMT3, F_1,20=1.31, P=0.27; DNMT4, F_1,19=0.06, P=0.82; DNMT5, F_1,20=1.46, P=0.24; DNMT6, F_1,20=0.58, P=0.44; DNMT7, F_1,20=1.20, P=0.29; GNMT, F_1,19=3.49, P=0.08; TET1, F_1,20=1.58, P=0.22; TET2, F_1,20=0.94, P=0.34; TET3, F_1,20=1.83, P=0.19) (Table 3).

Interest in understanding how parental generations influence their offspring phenotypes – essentially, epigenetic inheritance – has burgeoned in the past decade (Bautista and Burggren, 2019; Bhandari et al., 2015; Bošković and Rando, 2018; Nilsson et al., 2018; Skvortsova et al., 2018). Although transgenerational inheritance occurs from both parental lines, demonstration of the influence of the parental generation on offspring fitness and survival has typically used experimental designs testing only one parental line at a time (Lombó et al., 2015; Nye et al., 2007), and usually focusing on maternal effects (Green, 2008; Ma et al., 2018; Vega-Trejo et al., 2018). We exposed various combinations of female and male fish to crude oil extracts via diet, within environmentally relevant oil concentrations (Vignet et al., 2014), and derived F₁ larval crosses that allowed testing of the separate contributions of the maternal and paternal inheritance lines on offspring. Although oil exposure produced no phenotypic differences in the parental generation (i.e. growth, hypoxia tolerance nor maximum heart rate), both of the epigenetic markers studied (histone modification and global DNA methylation) were modified. In the offspring, survivability during crude oil exposure was differentially influenced by inheritance from both parents. Additionally, larval epigenetic regulation in the control condition was also influenced by both parental lines. Our results highlight the importance of the maternal and paternal exposure experience for offspring survivorship during crude oil exposure (and perhaps other stressors in early development).

Studies investigating the effects of crude oil exposure on adult fish are scarce and have been overshadowed by those focusing on early life stages (Pasparakis et al., 2019). However, studies on juvenile and young adult mahi-mahi (Coryphanea hippurus), as well as red drum (Sciaenops ocellatus), indicate that exposure to crude oil via water decreases swimming performance and metabolic rate (Johansen and Esbaugh, 2017; Mager et al., 2014; Stieglitz et al., 2016). Additionally, at the tissue and organ level, reduction in cardiac output occurs after crude oil exposure in juvenile fish, potentially resulting from compromised cardiomyocyte contractility by deceleration of the repolarization current and reducing the influx of Ca²⁺ into the cardiomyocytes, leading to prolonged action potentials (Brette et al., 2017; Heuer et al., 2019; Nelson et al., 2016).

In the present study, dietary exposure to a sublethal concentration of crude oil for 3 weeks did not compromise survivorship, growth or hypoxia tolerance of the adult population. These results suggest that the adult fish were in a healthy state during experimentation (Mauduit et al., 2016), and are further supported by the lack of difference in heart rate and tissue-specific somatic indices among groups. Noteworthy is the fact that MS222 acts as a cardio depressant, and some studies suggest that the use of this agent to induce anesthesia affects experimental results (Huang et al., 2014, 2010), thus masking differences among experimental groups. However, those effects have been seen when using doses (i.e. 160–200 mg l⁻¹) well above those used in the present study (90 mg l⁻¹). Therefore, we consider our results to be valid, and confirm that the dietary crude oil exposure levels used did not induce differences among the groups and, thus, the fish were in a healthy state (Mauduit et al., 2016).

No differences in adult phenotype were observed in the present study in response to dietary oil exposure, though some epigenetic regulation is suggested. The histone modification marker H3K4me3, which is associated with promoters of activated genes (Toni and Padilla, 2016), was less represented in the hearts of oil-exposed adults, suggesting downregulation of histone modified. Accordingly, oil-exposed adults had higher expression levels of H3K27me3, an epigenetic marker for inactive genes, compared with non-exposed adults. These results suggest that dietary oil exposure likely downregulates several chromatin regions in fish hearts. However, determining the specific outcome of this epigenetic modification and the targeted downregulated genes warrants further investigation.

In gonadal tissue, only oil-exposed males exhibited higher expression of both histone-related antibodies, suggesting some sex-specific global response to the environment, but not necessarily a specific change in chromatin regulation. The level of histone modifications in female gonads did not differ between the control and exposed individuals. A previous study on bisphenol A exposure revealed that this toxicant can downregulate oocyte maturation and, therefore, fish reproductive success through changes in the chromatin structure, mediated by an increase of H3K27me3 (Santangeli et al., 2016). The lack of changes in the gonads in the present study suggests that other epigenetic modifications, such as DNA methylation, could be more involved in the survivability of the offspring populations and also a previously observed decrease in adult zebrafish fecundity (Bautista and Burggren, 2019). However, these speculations warrant deeper investigation.

The decrease in global DNA methylation observed in cardiac tissue was accompanied by the upregulation of GNMT1, involved in the transfer of methyl groups from S-adenyl methionine (SAM) to glycine S-adenosylhomocysteine. This result aligns with that from a previous study in zebrafish in which exposure to 2.4 µg l⁻¹ of the PAH benzo(a)pyrene increased GNMT expression, which in turn decreased SAM supply and demethylation of the embryo (Fang et al., 2013). Although DNA methylation patterns are generally established early during embryogenesis (Fang et al., 2013), our results suggest that this epigenetic modification is highly plastic in response to environmental exposures that also occur at later life stages. Therefore, such epigenetic responses can occur at any point throughout the life time of organisms and, if adaptive, could allow individuals to rapidly cope with environmental stressors (Burggren, 2016; Heckwolf et al., 2019 preprint).

In contrast to cardiac tissue, global DNA methylation levels of the gonads were not affected by oil exposure. In addition, the transcription level of TET3, involved in demethylation and previously reported to affect DNA methylation levels in fish (Xiong et al., 2018), was downregulated. This indicates a possible modification of the methylation patterns in gonadal tissue as a response to oil exposure. Noteworthy is that we carried out the measurements in whole gonadal tissue, which holds a high level of cell heterogeneity, which might have masked the changes in global DNA methylation levels in the gametes. Therefore, changes in DNA methylation at specific sites (i.e. promoters) may still happen and cannot be ruled out (Falisse et al., 2018; Fang et al., 2013), which highlights the need of more in-depth research focused on addressing both this specific hypothesis and also the suitability of DNA methylation sequencing techniques.

Early developmental stages in fish are highly sensitive to environmental stressors, and their survival depends upon both their own growing capacities to cope with the environment as well as the phenotypes inherited from their parents (Auge et al., 2017; Burggren and Dubansky, 2018; Mager et al., 2014). Crude oil exposure in parental populations is mostly related to detrimental effects for their offspring (Corrales et al., 2014; Incardona and Scholz, 2018). However, depending upon the conditions, the inheritance of characters can be adaptive and may enhance the resistance against stressors or even increasing the niche width of the offspring (Bautista and Burggren, 2019; Herrera et al., 2012).

Most studies focused on determining the effects of crude oil on fish populations have reported detrimental effects for their offspring. Sometimes, those effects last up to three (and probably more) generations (Akhter et al., 2018; Corrales et al., 2014). Although we certainly acknowledge that crude oil exposure induces multiple effects on fish (reviewed in Pasparakis et al., 2019), our approach was to look for the existence of those characters that may actually be advantageous and enhance the survivability of the offspring, especially under the exposure to similar stressors to which the parental population experienced. Enhanced survivorship is, in our view, one of the clearest forms of evidence indicating that parental exposure to crude oil is having a positive effect on the offspring population (during exposure). This enhanced survivability may be due to transgenerational epigenetic inheritance from their parents. Therefore, some epigenetically inherited characters may be ‘adaptive’.

Offspring survival rates in the present study were differentially affected depending upon which parental sex had been exposed to oil. Thus, when raised in clean water, the offspring groups derived from exposed male adults (C♀E♂ and E♀E♂) exhibited lower survival in comparison to offspring from control males (C♀C♂ and E♀C♂). These results suggest that paternal exposure has an important role in offspring survival in clean water. Previous experiments on killifish embryos derived from parental groups where both of the parents were exposed to creosote contamination in their natural environment also reported lower survival in offspring raised in clean, non-polluted water (Meyer and Di Giulio, 2003). However, no information about the relative importance of each parent was analyzed. Nonetheless, in the present study, when offspring from oil-exposed parents (C♀E♂, E♀C♂ and E♀E♂) were exposed to crude oil via their ambient water, the three groups exhibited enhanced survival compared with controls. Similar results were found in killifish and zebrafish, where offspring from exposed parents were more resilient to the stressor to which the parents were exposed (Bautista and Burggren, 2019; Clark et al., 2014). The present findings highlight that both parental lines are involved in the regulation of survival capacities of their offspring when submitted to a stressor.

Parental oil exposure had no effect on offspring performance when F₁ larvae were challenged to LOE under hypoxia. When larvae were raised in clean water, the heart rate of offspring derived from parentally exposed groups (C♀E♂, E♀C♂ and E♀E♂) exhibited bradycardia in comparison to controls (C♀C♂). The major differences were seen after the hatching period (2 dpf) and align with the change from intrinsic to extrinsic control of cardiac function in this species (Lema et al., 2007; Schwerte et al., 2006). In addition, regardless of the nature of parental oil exposure, exposure to crude oil via their ambient water induced bradycardia in all experimental groups. Nonetheless, it still remains for additional studies to indicate whether the decrease in heart rate in larval fish is adaptive or maladaptive under stressful conditions (Farrell, 2007; Perry and Desforges, 2006).

Environmental exposures of both parents can lead to similar larval heart rate decreases. Consistent with these results, the relative color intensity levels of H3K4me3 in cardiac tissue from offspring derived from the parental exposed groups was higher in comparison with the control offspring. This suggests that, regardless of the parental line exposed to oil, this parental exposure can lead to the potential activation of regions and genes in the chromatin of their offspring even when these larvae are raised in clean water conditions. Remarkably, when both parents were exposed, the expression level of H3K4me3 was higher, suggesting an ‘additive’ effect. This result is further supported by the fact that H3K27me3 (associated with gene silencing) did not differ among groups.

Epigenetic responses in larval muscular tissue were somewhat similar to that in heart tissue. Offspring derived from the E♀C♂ and E♀E♂ groups exhibited higher color intensity levels of H3K4me3 than the C♀C♂ and C♀E♂ groups. This suggests that the maternal exposure condition may lead to the activation of genes in the muscle tissue of their offspring. In contrast, the C♀E♂ group exhibited higher relative color intensity of H3K27me3 compared to the control group (C♀C♂) and the E♀C♂ and E♀E♂ groups, supporting the idea that paternal oil exposure may lead to downregulation of genes in skeletal muscle. However, this effect may be somewhat offset by the maternal effect, because the E♀E♂ group was not different from the control or the E♀C♂ groups. The levels of these histone modifications in the offspring groups were thus tissue-specific and may be potentially related to specific functions, warranting further investigation.

Global DNA methylation in F₁ offspring in the present study was affected similarly by parental oil exposure. Methylation levels in the offspring derived from one or both parents exposed to crude oil were decreased in comparison to control offspring. Previous studies have suggested that pooling tissue may obfuscate global DNA methylation responses owing to tissue methylation heterogeneity (Cavalieri and Spinelli, 2017; Falisse et al., 2018). Our findings suggest that this may be specific to the experimental design and exposure conditions, and the interpretation may be influenced by the questions of interest. Noteworthy is that none of the transcript levels of genes involved in modifications of DNA methylation were affected by parental oil exposure in any of the larval groups, and along with the fact that CYP1A was also not upregulated, these results suggest that the change in DNA methylation observed in the offspring population is likely to be an epigenetically inherited character derived from the parental exposure to crude oil. In fish, although maternal DNA methylation levels are reset during early embryogenesis, the paternally inherited methylome is maintained constant (Ci and Liu, 2015; Jiang et al., 2013). This suggests that maternal inheritance will lead to responses in the offspring that would be different compared to the responses triggered by the paternal side. Although more specific studies of methylation patterns at specific sites (such as gene promoters) are needed to elucidate the specific role of DNA methylation on gene regulation, determination of global DNA methylation levels may provide early insight into transgenerational epigenetic effects.

The present study demonstrated differential upregulation of GATA4 and NPPA (genes involved in cardiac development) by paternal oil exposure and downregulation of NKX2.5 (gene involved in cardiac stress) by maternal oil exposure. This suggests that the parental exposure could, indeed, translate into changes in regulation of cardiac function in the F₁ offspring. This finding could explain in part the decreased heart rate observed in the early-stage offspring even when raised in clean water. Determining whether differential functional regulation is the result of histone modifications, changes in DNA methylation of gene promoter regions, or another molecular regulatory mechanism will require additional investigation.

Interest in the specific, differential roles of the maternal and paternal influence in offspring performance is rapidly increasing as researchers move beyond the default position that effects are via the maternal line. Although differential inheritance of phenotypic traits from male and female ancestors has been speculated to involve epigenetic mechanisms, empirical evidence addressing this phenomenon is scarce, partially owing to the complexities of designing definitive experiments (Akhter et al., 2018). In this study, we demonstrate that crude oil exposure is an epigenetic modifier for both parental lines, which are differentially involved in offspring inheritance and responses. Finally, more studies should focus on understanding the particular role of methylation patterns at the CpG islands of promoters or in the gene body per se, which may cause stable transcriptional gene silencing or enhanced expression, respectively (Cavalieri and Spinelli, 2017). In this process, epigenetic factors are essential to respond to rapidly changing environmental conditions, especially when multiple stressors occur in various combinations. Consequently, studies considering more than one stressor at a time should be particularly informative.

Results and figure legends in this paper are reproduced from the PhD thesis of Naim M. Bautista (Department of Biological Sciences at the University of North Texas, 2019).