Effects of indigowoad root (Radix Isatidis) on the immune responses and HSP70 gene expression of medicinal leeches (Poecilobdella manillensis) under Proteus mirabilis infection

• Radix Isatidis has the potential to increase the immune and anti-oxidative properties of leeches.
• Radix Isatidis has the potential to increase resistance to bacterial pathogens.
• Radix Isatidis has the potential to affect the expression of HSP70 in leeches.

We aimed to assess the effect of Radix Isatidis dietary supplementation on resistance to Proteus mirabilis infection in Poecilobdella manillensis. Six groups of leeches housed in tanks were randomly divided into five treatment groups (fed a basal diet supplemented with 2.5, 5.0, 7.5, 10.0, or 12.5 g/kg of Radix Isatidis) and one control group (fed a basal diet). Compared with the control group, the specific growth rate (SGR) and weight gain (WG) were significantly decreased in the treatment groups supplemented with Radix Isatidis. However, supplementation with 2.5 to 10.0 g/kg of Radix Isatidis did not produce an effect on mortality. Mortality was recorded after the leeches were challenged with P. mirabilis, and changes were observed in the serum cortisol, glucose, alanine aminotransferase (ALT), 3,5,30-triiodothyronine (T3), thyroxin (T4), alkaline phosphatase (ALP), total protein (TP), globin, and lysozyme contents. In the ingluvies, changes were observed in the total anti-oxidative capacity, superoxide dismutase (SOD) and catalase (CAT) activity, malondialdehyde (MDA) content and heat shock protein 70 (HSP70) gene expression over a period of 7 d. Treatment with 5.0 to 10.0 g/kg of Radix Isatidis increased the following parameters: total anti-oxidative capacity of the ingluvies prior to infection and at 1, 2 and 7 d after infection; serum ALP activity and serum globin concentrations prior to infection and at 1 and 2 d after infection; T4 concentrations prior to infection; serum lysozyme activity at 1 d after infection; SOD and CAT activity in the ingluvies prior to infection and 7 d after infection; T3 concentrations at 1 and 2 d after infection; and the relative level of HSP70 mRNA expression in the ingluvies at 2 d after infection. However, treatment with 5.0 to 10.0 g/kg of Radix Isatidis increased the following parameters: ALT contents prior to infection and 1, 2 and 7 d after infection; serum cortisol concentrations and serum glucose at 1, 2 and 7 d after infection; MDA contents of the ingluvies at 1 and 2 d after infection; and T4 concentrations at 2 and 7 d after infection. Compared with the control group, significantly lower mortality was observed in the groups supplemented with 5.0 to 10.0 g/kg of Radix Isatidis. These results suggest that supplementation with 5.0 to 10.0 g/kg of Radix Isatidis in the basal diet could enhance resistance to P. mirabilis infection in P. manillensis.Statement of relevanceWe believe that this manuscript is appropriate for publication in Aquaculture because it provides a natural method of controlling disease in P. manillensis, thereby avoiding the use of antibiotics and the development of antibiotic resistance. Furthermore, the results of our study provide new information on the physiological and molecular mechanisms that underlie the protective effect of Radix Isatidis.