Abstract:Mytilus coruscus, an economically important cultured shellfish in China, undergoes settlement and metamorphosis during its transition from planktonic to benthic life. The settlement of marine invertebrates is regulated by microbial biofilms. Bacteria and diatoms, along with their secreted extracellular polymeric substances, adhere to the substrate's surface. Diatoms can form biofilms independently of bacteria and are preferred by settling marine invertebrates. Previous studies extensively investigated, the mechanisms by which bacteria regulate the settlement of invertebrates. However, the relationship between diatom settlement and marine invertebrates, such as mussels, has yet to be reported. Gouqi Island, in Zhoushan, Zhejiang Province features open near-shore waters, and its surrounding area constitutes an important marine region for the raft culture of M. coruscus. This study aimed to investigate the interactions between diatoms and marine invertebrates and evaluate the potential of diatom biofilms as settlement inducers for juvenile mussels in M. coruscus. Specifically, the relationship between the diatom community in biofilms with varying ages formed at Gouqi Island and the settlement of M. coruscus was examined. Natural biofilms were established for 7, 14, 21, and 28 days in the marine area surrounding Gouqi Island (122°46'E; 30°43'N). Ten juveniles were placed in sterilized glass dishes (64 mm × 19 mm) with 20 mL of autoclaved filtered seawater and natural biofilms to test the inducing effect of biofilms on M. coruscus settlement. The biological characteristics, including dry weight, bacterial density, diatom density, and chlorophyll a content, of biofilms were analyzed. The population composition was identified by 18S rRNA amplicon sequencing (573F CGCGGTAATTCCAGCTCCA, 951R TTGGYRAATGCTTTCGC) and scanning electron microscopy. On average, 90% of juvenile mussels settled on 21- and 28-day biofilms. Results indicated a positive correlation between and factors such as biofilm age, diatom density, chlorophyll a concentration, dry weight, and bacterial density. The correlation between diatom density and juvenile mussel settlement was the strongest, with a coefficient of R = 0.96. Furthermore, the correlation between diatoms and juvenile mussel settlement was significantly higher than that observed for bacterial density, which had a correlation coefficient of R = 0.88. The dry weight of biofilms significantly increased with age, reaching 5.91 mg at 28 days. Bacterial density also increased over time, peaking at 2.68×106 cell/cm2 . The concentration of chlorophyll a exhibited a trend similar to that of diatom density, reaching its peak at 21 days. Although a decreasing tendency was observed at 28 days, the difference in concentration compared with that at 21 days was not statistically significant. The results of 18S rRNA sequencing showed significant differences (P ＜ 0.05) in eukaryotic microorganism diversity among different biofilm groups. The 18S rRNA sequencing analysis indicated that diatom phyla were predominant in biofilms of varying ages, exhibiting an average relative abundance of 0.96. The identified species within the diatom phyla encompassed 10 phyla, 4 orders, 17 families, 23 genera, and 32 species. The top 5 genera in terms of abundance in the sample were Licmophora, Odontella, Halamphora, Navicula, and Pauliella. The settlement of juvenile mussels positively correlated with the abundance of diatoms. Among the top 16 genera, the abundance of Thalassiosira, Licmophora, Minutocellus, and Achnanthes exhibited higher positive correlations with the settlement of juvenile mussels. Except for Licmophora, the abundance of Thalassiosira, Minutocellus, and Achnanthes showed higher relative abundance in the high biofilm day-age group than in the low biofilm day-age group. The relative abundances of Seminavis and Nitzschia negatively correlated with the settlement of juvenile mussels, and they were greater in the low biofilm day-age group than in the high biofilm day-age group. The relative abundance of diatoms with inducing activity for invertebrate settlement, such as Achnanthes, was higher in the high-age biofilms than in the low-age biofilms, whereas non-inducing diatoms such as Nitzschia had higher relative abundance in the low-age biofilms than in the high-age biofilms. The types and relative abundance of diatoms with and without inducing activity in biofilms were correlated with the induction capacity of these biofilms. This study indicated that diatoms may exhibit a higher inducing activity for invertebrates’ settlement, and the inducing activity may be related to their ecological habits, living environment, and physiological characteristics. The findings of this study provide a theoretical basis for examining the relationship between biofilm diatom community diversity and invertebrate settlement. This research offers a new perspective on addressing the challenge of seedling settlement during mussel aquaculture. Additionally, it has significant implications for applying diatoms and their biofilms to enhance shellfish aquaculture efficiency, particularly for M. coruscus. The insights from this study are crucial for understanding how to effectively utilize diatoms and their biofilms in cultivating M. coruscus.
