In Vitro Culture and Comparative Pathogenic Characteristics of Two Ciliates Prevalent in the Sea Cucumber Apostichopus Japonicus

Abstract

Ciliate diseases have become a significant concern in sea cucumber culture, yet their pathogenic mechanisms remain largely understudied due to the difficulty of continuous in vitro culturing. This study aimed to develop a long-term in vitro culture method for ciliates, so as to investigate their proliferation and pathogenic mechanisms at the molecular level. Hence, a novel “ciliate-primary cell co-culture in vitro” method was established firstly, to study the proliferation and pathogenicity of two main prevalent ciliates, Boveria labialis (B. labialis) and Uronema marinum (U. marinum), in Apostichopus japonicus (A. japonicus). This method allowed the facultative parasite U. marinum to proliferate continuously in vitro for over 30 months and enabled the obligate parasitic B. labialis to survive for 65 days at 4 oC, significantly longer than previously reported. The results demonstrated that the proliferation characteristics and pathogenicity of the two ciliates differ markedly in A. japonicus. B. labialis was found obligately parasite on the respiratory tree of A. japonicus, with a parasitic rate of 88.3% (544/616), showing no significant difference between healthy and diseased sea cucumbers. No proliferation behavior was observed in in vitro culture. In naturally growing sea cucumbers, B. labialis was found both sexually and asexually for proliferation. In contrast, U. marinum was isolated from the intestines of all six diseased sea cucumbers and was absent in healthy sea cucumbers. When in vitro co-culture with respiratory tree, body cavity, and intestinal cells, U. marinum proliferated rapidly mainly through binary fission. It had the highest proliferation rate in intestinal cells, which increased from 10 ciliates/mL to 2.56×104 ciliates/mL in 4 days. When the cultured ciliates were injected into healthy A. japonicus, 100% of the sea cucumbers became diseased. Pathological analysis indicated that U. marinum caused lesions in almost all organs, including epidermal ulcers and tube foot shedding, underscoring its high pathogenicity and potential threat to sea cucumber culture. This study enhances our understanding of ciliate pathogenic mechanisms and is vital for developing control strategies against ciliate diseases in aquaculture.

Keywords: Ciliate, in vitro co-culture, proliferation characteristics, pathogenic mechanism, Apostichopus japonicus