Development of an experimental protocol for the production of gnotobiotic clams

ABSTRACT Given the current scenario of global warming, which results in extreme atmosphere events (such as drought , floods, sea level rise, ocean acidification, increase in hypoxia), it is clear that climate change is a threat on aquatic and marine biota, fisheries and aquaculture. In this regard, one of the major concerns is linked to Heat Waves (HW), whose frequency and intensity are rising. This study is focused on Manila Clam (Ruditapes philippinarum), the second most important bivalve species in fisheries and aquaculture. Prolonged periods of high temperature have been proven to significantly affect R. philippinarum by decreasing survival rate, growth, and reducing their genetic variability. Clams’ physiology is largely affected by their microbial communities, collectively called ‘microbiota’, which play a key role in clam growth, health and protection from pathogenic infections. Recent studies suggested that alteration o fclam microbiota is one of the first responses to environmental changes, such as heat waves and pollution. Nevertheless, the potential adaptationl of clam’s microbiota to climate change and the effect of such process on clams’ physiology remain elusive. In this frame, generation of gnotobiotic animals is crucial to understand the mechanisms underlying clam’s microbiota function and adaptation. The aim of this study is to develop the first protocol to generate Germ-Free clams in order to ultimately create gnotobiotic animals. A set of different variables (i.e., type of water, antibiotic type, concentration, and timing of inoculum, animal cleaning procedures, etc.) has been tested to optimize the conditions for generating and maintaining GF clams. Animals were sacrificed and analyzed at different time points on general and selective microbiological media to monitor bacterial presence and growth. The developed experimental protocol, which involved the use of deionized water, supplemented with salt, a mix of six antibiotics (ampicillin, tetracycline, kanamycin, penicillin and cycloheximide), led to a 5-log reduction of the initial clam bacterial load (from 107 CFU/ml to 102 CFU/ml).The microbial community that persisted after antibiotic treatment was analysed by 16S DNA gene sequencing, with V3-V4 primers, on the Illumina MiSeq platform.The developed protocol will serve to transfer defined microbial communities into clam hosts, among which microbial communities potentially adapted to heat stress, and test their effect on host physiology. This will allow disclosing the role of clam microbiota on host response to climate change and the potential beneficial effect of adapted microbial communities.

ABSTRACT Given the current scenario of global warming, which results in extreme atmosphere events (such as drought , floods, sea level rise, ocean acidification, increase in hypoxia), it is clear that climate change is a threat on aquatic and marine biota, fisheries and aquaculture. In this regard, one of the major concerns is linked to Heat Waves (HW), whose frequency and intensity are rising. This study is focused on Manila Clam (Ruditapes philippinarum), the second most important bivalve species in fisheries and aquaculture. Prolonged periods of high temperature have been proven to significantly affect R. philippinarum by decreasing survival rate, growth, and reducing their genetic variability. Clams’ physiology is largely affected by their microbial communities, collectively called ‘microbiota’, which play a key role in clam growth, health and protection from pathogenic infections. Recent studies suggested that alteration o fclam microbiota is one of the first responses to environmental changes, such as heat waves and pollution. Nevertheless, the potential adaptation of clam’s microbiota to climate change and the effect of such process on clams’ physiology remain elusive. In this frame, generation of gnotobiotic animals is crucial to understand the mechanisms underlying clam’s microbiota function and adaptation. The aim of this study is to develop the first protocol to generate Germ-Free clams in order to ultimately create gnotobiotic animals. A set of different variables (i.e., type of water, antibiotic type, concentration, and timing of inoculum, animal cleaning procedures, etc.) has been tested to optimize the conditions for generating and maintaining GF clams. Animals were sacrificed and analyzed at different time points on general and selective microbiological media to monitor bacterial presence and growth. The developed experimental protocol, which involved the use of deionized water, supplemented with salt, a mix of six antibiotics (ampicillin, tetracycline, kanamycin, penicillin and cycloheximide), led to a 5-log reduction of the initial clam bacterial load (from 107 CFU/ml to 102 CFU/ml).The microbial community that persisted after antibiotic treatment was analysed by 16S DNA gene sequencing, with V3-V4 primers, on the Illumina MiSeq platform.The developed protocol will serve to transfer defined microbial communities into clam hosts, among which microbial communities potentially adapted to heat stress, and test their effect on host physiology. This will allow disclosing the role of clam microbiota on host response to climate change and the potential beneficial effect of adapted microbial communities.