During the first year, robust laboratory culturing conditions for the target marine bristleworms were established, and a targeted field sampling campaign was successfully completed to collect the specimens necessary to sustain the experimental design. An extensive sampling effort covered seven critical developmental transitions for both target species, yielding four to six biological replicates per stage. High quality RNA was extracted from 158 distinct samples for RNA sequencing. Bioinformatic analyses are actively identifying differentially expressed genes to generate transcriptional profiles that describe species-specific developmental trajectories and to identify the complex transcriptional changes underlying their divergent developmental and reproductive dynamics (WP1). These findings are currently in preparation for publication in peer reviewed journals. In the second year, the project deployed a state-of-the-art naturalistic environmental light intensity system. This allowed the simulation of sunlight and moonlight spectra, including intensity changes associated with the rotation of the Earth and the revolution of the Moon, exactly as perceived at about 5 meters below the sea surface. Using these advanced environmental simulations, the project successfully mapped the behavioral responses (reproductive rhythms) of both species to naturalistic moonlight, and is currently in the process of unveiling the underlying molecular dynamics. For this, the project established a highly detailed temporal sampling over the course of multiple lunar cycles to reveal and profile how these two species process lunar cues, findings that are currently in preparation for publication in peer reviewed journals. To achieve this, tissues were sampled at high resolution every three to four days across fully entrained lunar months and lunar free-running months to discriminate between environment driven and endogenous driven molecular dynamics. Additionally, a global network of collaborators was established to collect fixed specimens worldwide. The project extracted, sequenced, and molecularly identified about 700 DNA barcoding sequences to resolve population structure and update the phylogeny of the genus. In terms of exploitation and dissemination, the project actively engaged the general public through outreach events featuring custom designed educational board games to enhance social knowledge on evolution and marine biodiversity. Scientifically, molecular and morphological datasets will be made openly accessible, and the findings will be disseminated through international conferences.
