Periodic Reporting for period 1 - AcidICC (Understanding ocean Acidification Impacts on Chemical Communication in marine species)
Reporting period: 2022-01-01 to 2023-12-31
Ocean acidification (OA) has major consequences on marine ecosystems. Survival of species and particularly calcifying species was shown to be decreased at lower pH as the latter directly damages their protective shells. However, the impacts of OA on chemically mediated behaviors have often not been considered or studied. Chemical cues are of major importance for several categories of behavior in marine species: finding habitat/prey/mating partners and avoiding predator. If chemical communication is disrupted, organisms might not find their mating partners or synchronize the release of gametes (in case of spawning species), the reproduction will not occur and the lineages and even the species could in the long-term be extinct.
Acidicc project investigated the impacts of OA on chemical communication in the polychaete Platynereis dumerilii. P. dumerilii is a promising species to investigate the impact of OA on chemical communication and reproduction. Indeed, this new model, is easy to culture, is globally distributed, its genome is sequenced, and its pheromones involved in its mating behavior have been mostly identified. When mature, the males and females become pelagic and reproduce at night (after specific lunar cycle). As broadcast spawner, P. dumerilii males and females use pheromones (5-methyl-3-heptanone, uric acid, ovothiol A, 5-octadiene-2-one) to induce rapid swim speed or to trigger the release of their gametes synchronously.
In this study, we investigated the impacts of OA on the chemical communication process at different steps: alteration of the cue itself or its production, reception of the cue and resulting behavior. The Objective 1 of AcidICC was to quantify the uric acid production in P. dumerilii females from different pH environments, at a lower pH with higher pH fluctuations. By doing so we could determine if the pH influences the production of uric acid and if the females acclimatize to low pH. Objective 2 aimed to quantify the cue detection response in P. dumerilii males from different pH environments at lower pH with higher pH fluctuations, and measure the potential costs of pH adaptation or acclimatization (e.g. survival) in P. dumerilii males and females. Since nothing was known about the genes coding for P. dumerilii pheromone, we aimed to find candidate genes likely coding for the receptor of the sperm release pheromone (uric acid) using comparative transcriptomic and GPCR deorphanization to test ligand activation of candidate GPCRs (Objective 3). Then, Objective 4 was to carry knockout of such candidates using CRISPR/Cas-9 technology to test if the candidate genes are indeed coding for the receptor, and in situ hybridization to detect where it is expressed. Once identified, the Objective 5 aims to determine the conformation changes of such receptors and of the pheromone under different pHs. Finding the receptor is important to investigate the effect of pH on an identified receptor-ligand pair and to genetically dissect the function of the receptor during spawning.
Acidicc project investigated the impacts of OA on chemical communication in the polychaete Platynereis dumerilii. P. dumerilii is a promising species to investigate the impact of OA on chemical communication and reproduction. Indeed, this new model, is easy to culture, is globally distributed, its genome is sequenced, and its pheromones involved in its mating behavior have been mostly identified. When mature, the males and females become pelagic and reproduce at night (after specific lunar cycle). As broadcast spawner, P. dumerilii males and females use pheromones (5-methyl-3-heptanone, uric acid, ovothiol A, 5-octadiene-2-one) to induce rapid swim speed or to trigger the release of their gametes synchronously.
In this study, we investigated the impacts of OA on the chemical communication process at different steps: alteration of the cue itself or its production, reception of the cue and resulting behavior. The Objective 1 of AcidICC was to quantify the uric acid production in P. dumerilii females from different pH environments, at a lower pH with higher pH fluctuations. By doing so we could determine if the pH influences the production of uric acid and if the females acclimatize to low pH. Objective 2 aimed to quantify the cue detection response in P. dumerilii males from different pH environments at lower pH with higher pH fluctuations, and measure the potential costs of pH adaptation or acclimatization (e.g. survival) in P. dumerilii males and females. Since nothing was known about the genes coding for P. dumerilii pheromone, we aimed to find candidate genes likely coding for the receptor of the sperm release pheromone (uric acid) using comparative transcriptomic and GPCR deorphanization to test ligand activation of candidate GPCRs (Objective 3). Then, Objective 4 was to carry knockout of such candidates using CRISPR/Cas-9 technology to test if the candidate genes are indeed coding for the receptor, and in situ hybridization to detect where it is expressed. Once identified, the Objective 5 aims to determine the conformation changes of such receptors and of the pheromone under different pHs. Finding the receptor is important to investigate the effect of pH on an identified receptor-ligand pair and to genetically dissect the function of the receptor during spawning.
While studying the impacts of lower pH on the cues themselves (O1), we found that lower pH impacts the protonation stage of the 5-methyl-3-heptanone and likely impacts the production of uric acid in females. For O1 and O2, we cultured worms at normal (8.1) and lower pH (7.7) we did preliminary behavioral assays using males from the different conditions and measured their swim speed after addition of uric acid. We also measured the quantity of uric acid being produced by females at different pH (8.1; 7.8) using a colorimetric quantifying test. These experiments need higher replicates number.
The receptor of uric acid is known to be in the modified parapodia of sexually matured P. dumerilii males. We performed comparative transcriptomics of modified and unmodified parapodia from males and females, in order to find candidate genes likely coding for this receptor (O3). We tested 35 candidate genes carrying out GPCR deorphanisation assays (O3). We successfully deorphanize 3 receptors with monoamines. None of the candidate receptors seemed to respond to uric acid. However, we found two candidate receptors responding to P. dumerilii pheromones, other than uric acid. Using HCR in situ hybridization (O4), we showed that these receptors are expressed in the dorsal part of the modified parapods in matured P. dumerilii males and females. We further characterized the structure where they are expressed with SEM imaging. With gene tree analyses, we explore the evolution of such GPCR new gene family. We will have to carry out knock out experiments to confirm the function of these receptors and then study their conformation un different pH conditions.
In parallel to these objectives, Dr. Moris participated to one TREC expedition in Faro in order to help finding P. dumerilii samples. She also took part in the study of the growth hormone of P. dumerilii. This publication is currently under review while two other publications regarding O3-O4 are in prep. She presented her results at three European/international conferences: EuroEvoDevo in Naples in 2022, ISCE meeting in Bangalore 2023, Polycheate meeting in Stellenbosch in 2023.
The receptor of uric acid is known to be in the modified parapodia of sexually matured P. dumerilii males. We performed comparative transcriptomics of modified and unmodified parapodia from males and females, in order to find candidate genes likely coding for this receptor (O3). We tested 35 candidate genes carrying out GPCR deorphanisation assays (O3). We successfully deorphanize 3 receptors with monoamines. None of the candidate receptors seemed to respond to uric acid. However, we found two candidate receptors responding to P. dumerilii pheromones, other than uric acid. Using HCR in situ hybridization (O4), we showed that these receptors are expressed in the dorsal part of the modified parapods in matured P. dumerilii males and females. We further characterized the structure where they are expressed with SEM imaging. With gene tree analyses, we explore the evolution of such GPCR new gene family. We will have to carry out knock out experiments to confirm the function of these receptors and then study their conformation un different pH conditions.
In parallel to these objectives, Dr. Moris participated to one TREC expedition in Faro in order to help finding P. dumerilii samples. She also took part in the study of the growth hormone of P. dumerilii. This publication is currently under review while two other publications regarding O3-O4 are in prep. She presented her results at three European/international conferences: EuroEvoDevo in Naples in 2022, ISCE meeting in Bangalore 2023, Polycheate meeting in Stellenbosch in 2023.
Acidicc project revealed a new GPCR family responding to P. dumerilii pheromones. The discovery of this new family of receptors is a significant since so far hardly anything was known about chemoreceptors in annelids. This exciting new discovery will contribute to developing Platynereis into a new molecular genetic model for chemical ecology. This is important as the systematic study of the impacts of ocean acidification in an experimentally tractable laboratory model species such as P. dumerilii should help to better predict how ocean acidification impacts the marine communities by modifying their chemical communication, whether acclimation and adaptation occur and at which costs.