The ongoing development of new production methods may lead to the commercialization of N-acetyl chitooligosaccharides (NACOS), such as chitosan oligosaccharides (COS). The bioactivity of NACOS, although not well detailed, differs from that of COS, as they have more acetyl groups than COS. We used two enzymatically produced NACOS with different molecular compositions and six NACOS (NACOS1–6) with a single degree of polymerization to verify their immunomodulatory effects on the RAW264.7 macrophage cell line. We aimed to identify any differences between COS and various NACOS with a single degree of polymerization. The results showed that NACOS had similar immune enhancement effects on RAW264.7 cells as COS, including the generation of reactive oxygen species (ROS), phagocytotic activity, and the production of pro-inflammation cytokines (IL-1β, IL-6, and TNF-α). However, unlike COS and lipopolysaccharide (LPS), NACOS1 and NACOS6 significantly inhibited nitric oxide (NO) production. Besides their immune enhancement effects, NACOS also significantly inhibited the LPS-induced RAW264.7 inflammatory response with some differences between various polymerization degrees. We confirmed that the NF-κB pathway is associated with the immunomodulatory effects of NACOS on RAW264.7 cells. This study could inform the application of NACOS with varying different degrees of polymerization in human health.

Cryptocaryon irritans is an obligate ectoparasitic ciliate pathogen of marine fishes. It can infect most marine teleosts and cause heavy economic losses in aquaculture. There is currently no effective method of controlling this disease, and little information is available regarding the genes involved in its development and virulence. We aimed to investigate the distinct features of the three major life-cycle stages of C. irritans in terms of gene transcription level, and identify candidate vaccines/drug targets. We established a reference transcriptome of C. irritans by RNA-seq. Three cDNA libraries using total poly(A)+ mRNA isolated from trophonts, tomonts, and theronts was constructed and sequenced, respectively. Clean reads from the three stages were de novo assembled to generated unigene. Annotation of unigenes and transcriptomic comparison of three stages was performed. Totals of 73.15, 62.23, and 109.57 million clean reads were generated from trophont, tomont, and theront libraries, respectively. After de novo assembly, 49,104 unigenes were obtained, including 9,253 unigenes with significant similarities to proteins from other ciliates. Transcriptomic comparisons revealed that 2,470 genes were differentially expressed among the three stages, including 2,011, 1,404, and 1,797 genes that were significantly differentially expressed in tomont/theront, tomont/trophont, and theront/trophont pairwise comparisons, respectively. Based on the results of hierarchical clustering, all differentially expressed genes (DEGs) were located in five major clusters. DEGs in clusters 1 and 2 were more highly expressed in tomonts than in other stages, DEGs in cluster 3 were dominant in the tomont and trophont stages, whereas clusters 4 and 5 included genes upregulated in the theront stage. In addition, Immobilization antigens (I-antigens) and proteases have long been considered major targets for vaccine development and potential drug targets in parasites, respectively. In the present study, nine putative I-antigens transcripts and 161 protease transcripts were found in the transcriptome of C. irritans. It was concluded that DEGs enriched in tomonts were involved in cell division, to increase the number of theronts and ensure parasite continuity. DEGs enriched in theronts were associated with response to stimuli, whereas genes enriched in trophonts were related to nutrient accumulation and cell growth. In addition, the I-antigen and protease transcripts in our transcriptome could contribute to the development of vaccines or targeted drugs. Together, the results of the present study provide novel insights into the physiological processes of a marine parasitic ciliate.