Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2025.12.16.694564

Methylphenidate exposure alters brain gene expression and induces transgenerational DNA methylation changes in Poecilia reticulata guppies

Alfaro, R. J.·De Serrano, A. R.·Sokolowski, D.·Hughes, K. A.·Rodd, F. H.·Anreiter, I.
View original source ↗Export metadata ↓

Abstract

Chronic exposure to stimulants is known to affect behavioral phenotypes and epigenetic profiles intergenerationally. We have previously shown that chronic methylphenidate hydrochloride (MPH) exposure in male guppies (Poecilia reticulata) induces persistent, paternally transmitted behavioral changes across multiple unexposed generations. Here, we investigated the underlying epigenetic signatures of this transgenerational behavioral inheritance. Building on our previous study, which used composite behavioral scores, we investigates the transgenerational inheritance patters of individual behaviors and found a robust, male-lineage-dependent increase in swimming across unexposed G2-G4 female offspring. Our molecular analysis showed that that chronic MPH exposure in G1 males alters brain gene expression, with 76 differentially expressed genes including genes with developmental and locomotory functions, and an over-representation of long non-coding RNAs. Furthermore, we found that unexposed G4 descendants from MPH-treated lineages exhibit widespread changes in brain DNA methylation, including genes related to developmental and swimming behavior. Interestingly, we found one lnRNA, LOC103476631, as both differentially expressed in G1 males and differentially methylated in G4 fish, providing a compelling candidate for ncRNA-directed DNA methylation as a mechanism for the observed transgenerational epigenetic inheritance of swimming behavior. Author SummaryDrugs used to treat attention-deficit/hyperactivity disorder, such as methylphenidate, are widely prescribed to children and adolescents during key stages of brain development. We previously showed that exposing male guppies to a low, chronic dose of methylphenidate changed anxiety-like behavior not only in the treated fish but also in several generations of their unexposed descendants. In this study, we asked how such long-lasting behavioral effects might be recorded in the brain. We first confirmed that a simple measure of swimming behavior remains altered in descendants of treated males across multiple generations. We then examined brains from the directly exposed fathers and from great-grand-offspring that were never exposed to the drug. In fathers, we found changes in the activity of genes, including many long non-coding RNAs, a class of genes that has been linked to transgenerational inheritance. In great-grand-offspring, we found significantly altered DNA methylation profiles, an epigenetic mark that has also been associated with transgenerational inheritance. Together, our findings suggest that developmental exposure to a commonly used stimulant can leave a stable molecular memory in the brain that persists across generations.

Explore related subjects

geneticsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Alfaro, R. J., De Serrano, A. R., Sokolowski, D., Hughes, K. A., Rodd, F. H., Anreiter, I.. 2025-12-18. Methylphenidate exposure alters brain gene expression and induces transgenerational DNA methylation changes in Poecilia reticulata guppies. https://doi.org/10.64898/2025.12.16.694564

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Integrated exome-transcriptome analysis of a schizophrenia case with an exceptionally low polygenic risk score

Schizophrenia liability reflects both common polygenic variation and rarer variants of larger effect. We investigated a post-mortem schizophrenia case (SZ07) with a schizophrenia polygenic risk score below the first percentile among ancestry-matched cases, asking whether individual-level genomic and transcriptomic analyses could help identify potentially novel schizophrenia-relevant variants. We used exome analysis with multiple variant-prediction tools to generate a panel of variants and corresponding candidate genes potentially implicated in the pathological process in SZ07. The prioritised variants were then assessed using RNA sequencing of three cortical regions. Of these, only an IZUMO4 splice-acceptor variant showed direct RNA-level support. Beyond variant-specific analyses, we characterised broader transcriptomic abnormalities in the SZ07 brain. Pathway analysis of gene-level expression deviations implicated innate immune processes, calcium signalling, synaptic biology, and axon remodelling. Notably, compared with conventional single-sample biological pathway analysis, the analysis we used showed only a weak association with neuronal specificity, suggesting their reduced sensitivity to the main axis of variation related to cellular composition in bulk brain tissue gene expression data. Although the candidate genes showed no significant pathway-level convergence, the broader transcriptomic profile revealed coherent biological alterations largely compatible with the documented history of cerebrovascular disease in SZ07. These results demonstrate how the integration of exome and transcriptome data at the individual level enables the formulation and constraint of hypotheses regarding patient-specific genetic features and their molecular consequences, while highlighting important limitations related to tissue composition, comorbidity, and the choice of reference cohort.

genetics↗
bioRxiv2026

A genetic screen to identify host factors required for commensal bacterial adherence to the gut epithelia

Bacterial adherence to gut epithelial cells likely involves host genetic components. We isolated a gut commensal bacterium, Lelliottia jeotgali, that can robustly colonize C. elegans through attachment to intestinal epithelial cells. Here, we developed a novel screen in C. elegans to identify host genes required for microbiome bacterial colonization. Using transgenic L. jeotgali strain expressing tdTomato, we conducted forward genetic screens in C. elegans to select for mutants that completely lost colonization. We identified multiple allelic mutations in five different C. elegans genes, termed knockout of Lelliottia adherence (kola) mutants, with a near complete loss of adherence. Each of kola-1-5 is uncharacterized and predicted to encode for a secreted or membrane-bound protein. We also identified gly-5, a GalNAc transferase that initiates O-linked glycosylation. Overall, we describe the first C. elegans screen to identify host factors that control microbiome bacterial colonization in the gut.

genetics↗
bioRxiv2026

Male-specific genome sequences for non-invasive sex identification in Nishikigoi (Cyprinus carpio)

Nishikigoi, ornamental cultivars of the common carp (Cyprinus carpio), are economically valuable aquaculture teleosts in which mature females command significantly higher market prices due to superior body conformation and coloration. Consequently, early and non-invasive sex identification is highly desirable for commercial breeding. Although sex determination in common carp follows an XX/XY system, sex-chromosome-specific markers and field-applicable diagnostic methods for Nishikigoi have remained limited. In this study, we conducted sequence-based bulked segregant analysis using whole-genome shotgun sequencing of female and male pools in Kohaku, a representative Nishikigoi lineage. We identified male-specific candidate contigs and successfully developed PCR-based male-specific markers. Validation across multiple Nishikigoi varieties and wild common carp demonstrated that primary primer pairs achieved high diagnostic accuracy. Comparative genomic analysis revealed that these candidate contigs mapped to the genomic region on chromosome 25B containing the male-determining gene Pangu, with the primary marker located in immediate physical proximity to the gene. To enable field diagnostics, we combined non-invasive epidermal mucus DNA extraction, loop-mediated isothermal amplification (LAMP), and chromatography-printed array strip (C-PAS) technology, establishing a rapid on-site detection workflow. Overall, this study provides highly reliable PCR markers associated with the sex-determination locus and establishes a non-invasive, field-applicable diagnostic platform to streamline commercial Nishikigoi breeding and sex-selected stock management.

genetics↗