Selected essential oil components fail to induce an immunological response in Artemia but still protect against vibriosis

In the present research, in order to screen out the best candidates from 12 different EOCs, we proposed three in vivo screening methods, namely the screening method of bioluminescence of V. campbellii associated with brine shrimp, regrowth performance of V. campbellii, and immune gene expression of brine shrimp without challenge. Our result showed that challenged with V. campbellii at 107 cells/mL, the survival of the brine shrimp at 48 h was significantly increased after treatment with the EOCs (at 0.0005%, v/v) of 4-allylanisole, R-(+)-limonene, S-(- )-limonene, (-)-terpinen-4-ol, (+/-)-citronellal, citral, trans-cinnamaldehyde and (+)-carvone, compared to the positive control group. Also, it was observed that the EOCs-of 4-allylanisloe, R-(+)-limonene, S-(-)-limonene, (- )-beta-pinene, geraniol, (+/-)-citronellal, citral, trans-cinnamaldehyde and (+)-carvone decreased significantly the in vivo bioluminescence of V. campbellii at 36 h after Vibrio exposure. The regrowth assay showed that inde-pendently from incubation time (1, 12 or 24 h), no difference was observed in the regrowth curve in all EOC treatment groups compared to the positive control group. The dscam gene expression in the (+/-)-citronellal group, and the sod gene in the citral group were observed to be significantly higher than in the negative control at 24 h, respectively. However, most of the immune genes were down-regulated in the EOC groups. Combining the survival data at 48 h with the bioluminescence result at 36 h, it was noted that the survival rate of brine shrimp was moderately correlated with in vivo bioluminescence of V. campbellii. The results indicate that the approach of determining in vivo bioluminescence of V. campbellii is a moderately reliable, fastest, and cheapest screening method for EOCs. As the regrowth performance assay of V. campbellii, and the immune genes expression assay of brine shrimp without challenge cannot predict Artemia survival properly, they cannot be used as screening methods for EOCs. Moreover, the immune genes expression assay is relatively slow, time-consuming and costly.

Automated summary

In this study, three in vivo screening methods were proposed to identify the best candidates from 12 different EOCs. The results showed that some EOCs significantly increased the survival rate of brine shrimp and reduced the in vivo bioluminescence of V. campbellii. The immune gene expression assay was not able to accurately predict the survival rate of brine shrimp and was time-consuming and costly.