Artificial Light at Night Disrupts Circadian and Metabolic Gene Expression in the Green Anole Lizard (Anolis carolinensis): A Transcriptomic Analysis

Artificial light at night (ALAN) disrupts natural light-dark cycles, posing ecological challenges for wildli in urban areas. Here we investigated the efcts of ALAN on gene expression in the brain, liver, skin, and gonads of green anole lizards (Anolis carolinensis) whose urban populations are increasingly exposed to light pollution. To identify genetic pathways impacted by ALAN exposure we analysed expression of genes associated with circadian and metabolic regulation at midday, midnight and at midnight with artificial light. Difrential expression analysis revealed that clock-related genes (PER1, NR1D1, CRY2) were significantly altered in the brain, liver, and skin following ALAN treatment and genes involved in glucagon regulation (GCG) and lipid metabolism (NOCT) were difrentially expressed in the liver, indicating metabolic disruptions. Skin exhibited unique responses to ALAN suggesting that repair responses may be altered as genes related to cellular processes, such as wound healing, were upregulated under normal light and dark conditions. Our findings also show that ALAN disrupts core circadian genes, impacting physiological processes including hormone regulation, glucose homeostasis, and potentially reproductive cycles. This study provides the first transcriptomic evidence of the efcts of light pollution on green anoles, highlighting the need to preserve natural light cycles in urban habitats. An interactive online database developed for this study allows further exploration of gene expression changes, to promote research on artificial light-polluted environments. Twenty-four free-living adult green anole lizards (Anolis carolinensis; twelve of each sex) were captured in the breeding season, in May 2024, on the urban campus of Trinity University in San Antonio, Texas, USA, during daylight hours. Green anole lizards were collected by using a dental floss loop attached to an extendable fishing pole or by hand and were transported individually to the Trinity University vivarium in cotton bags. On the day of capture, the body mass of each anole was measured to the nearest 0.1 g using a Pesola spring scale and snout-vent length measured to the nearest mm using a clear plastic ruler (Males: range 52-69mm, average 62mm; Females: range 51-57mm, average 55mm). Each individual was given a unique identification number on the lower jaw using a non-toxic permanent marker. Each anole was then randomly assigned to one of three treatment groups: Midday, Midnight, or ALAN. Sex was determined by the presence of hemipenes and the size of the dewlap; four anoles of each sex were assigned to each treatment group. Green anole collection was performed under Scientific Permit SPR-0310-045 to Michele A. Johnson from Texas Parks and Wildli Department, with approval from Trinity University’s Animal Research Committee, protocol 051122-MJ2.Green anoles were rapidly decapitated without prior anaesthesia to avoid any confounding anaesthetic efcts on RNA expression. Brain (containing the pineal gland), eyes, dorsal skin, ventral skin, liver, and testes or ovaries were collected in under 7 min. 33 s and flash frozen on dry ice and stored at -80 oC until they were shipped to BGI Genomics (San Jose, CA, USA). Midday treatment dissections were performed between 12:48 to 13:45, and Midnight and ALAN treatment from 22:30 to 00:42. Decapitation for the Midnight treatment group was performed in the dark under red torch light (HQRP, Harrison, NJ; emission peak in the red spectrum at 650 nm). To control for this additional illumination, ALAN treatment lizards were also illuminated by the same red torch light during decapitation.