The potential efficacy of plant extracts for abiotic-stress and disease prevention has received widespread attention in aquaculture. Few studies have been reported on the application of ELE in aquatic animal feeds. We reported the effects of dietary ELE on growth, muscle composition, non-specific immunity and diseases resistance of juvenile red claw crayfish. In the present study, a diet supplemented with 1–2 g kg− 1 ELE significantly increased the WGR and SGR of crayfish, which was similar with previous studies in weaned piglets (Peng et al., 2019) and broilers (Zhao et al., 2019). Supplementation with 4–10 g kg− 1 ELE did not significantly improve the growth performance of crayfish, which indicates that the beneficial effects of plant supplementation on crayfish growth are dose-dependent. If optimal doses are exceeded, the benefits might be lost (Reverter et al., 2021; Yousefi et al., 2021). The supplementation of excessive ELE in the feed could affect the metabolism of crayfish, leading to high energy consumption, stress and toxic effects on crayfish (Tan et al., 2017). Compared to the control group, dietary treatment of 1 g kg-1 ELE increased crude protein content and decreased crude lipid content in crayfish muscle. The results are consistent with previous studies on plant extracts (Sun et al., 2018; Tan et al., 2017). Sufficient nutrient utilization improves the physiological metabolism of crayfish, affecting the metabolism of amino acids, lipids and glycogen in the tissue. Crayfish fed diets with proper ELE levels alter protein and lipid utilization, causing lipid consumption and protein deposition. Our results indicated that dietary ELE could optimize the utilization of nutrients and increase protein synthesis in crayfish.
The hepatopancreas is an important organ in crustaceans and plays an important role in digestion, immune defense, nutrient absorption and metabolism (Ellis et al., 2011; Rombout et al., 2011). The nutritional status can be assessed by observing changes in the morphology of the hepatopancreas in crustaceans (Najdegerami et al., 2016; Raškovic´ et al., 2011). Previous studies reported that dietary ELE could significantly reduce an increase in lipid accumulation and preserve liver function in chickens and rats (Jin et al., 2013; Santoso, 2000). In this study, crayfish fed Diet 3 and Diet 4 showed regular hepatocyte morphology. However, hepatocytes of crayfish in the Diet 6 group showed cell swelling and mild vacuolation. The increased vacuolation of the crayfish hepatopancreas may be caused by the complex composition of ELE, suggesting that extra ELE may damage to the crayfish hepatopancreas, which may be the reason for not improving the growth performance of crayfish fed Diet 6 (Tan et al., 2018). Inadequate crayfish feed formulation may cause mild vacuolation of the hepatopancreas in the Diet 1 and Diet 2 groups. These results indicated that dietary supplementation with 1–2 g kg− 1 ELE diets could improve the morphology of the hepatopancreas with the improvement of absorptive function for various nutrients.
The antioxidant system plays a crucial part in protecting crayfish from oxidative stress (Yuan et al., 2016). Antioxidant enzymes are important components of the antioxidant system in crustaceans. The T-AOC refers to the antioxidant capacity of the enzymatic and non-enzymatic antioxidants of crustaceans (Xie et al., 2008). The SOD, GPx and CAT are important antioxidant enzymes of aquatic animals and can eliminate unnecessary free superoxide anion radicals (Ming et al., 2015; Tan et al., 2016). MDA can induce tissue damage, reflecting the degree of lipid peroxidation and cell damage (Muñoz et al., 2000). The results showed that T-AOC, GPx and SOD activities significantly increased, and the MDA content decreased in the hemolymph and the hepatopancreas of crayfish fed the optimal ELE level, which is similar to the findings in grass carp (Yang et al., 2021). Yang et al. reported that the supplementation of ELE in diets significantly increased the activities of SOD and GPx in grass carp (Yang et al., 2021). Previous studies reported that plant extract supplements could improve the antioxidant capacity of red swamp crayfish (Procambarus clarkii) (Cheng, 2019; Liu et al., 2020). The trends in the change expression levels of antioxidant genes were consistent with the enzyme activity analysis in this study. Our results showed that dietary ELE significantly up-regulated the expression levels of SOD, GPx and Se-GPx in the hepatopancreas of crayfish. Tan et al. reported that dietary Ginkgo (Ginkgo biloba) leaf extract could improve antioxidant ability in the head kidney of hybrid grouper (Epinephelus lanceolatus♂×Epinephelus fuscoguttatus♀) by increasing antioxidant gene expression (Tan et al., 2018). Consistent with these studies, our findings indicated that ELE supplementation in the diet could protect the hepatocytes and hemocytes from oxidative protein carbonation and lipid peroxidation by activating the antioxidant response in red claw crayfish (Liu et al., 2020).
Phosphatases are crucial enzymes in the biological processes of aquatic animals and shape immune responses and reflect the immune system in aquatic animals (Murti et al., 1984; Sarlin and Philip, 2011). ACP plays an essential role in substance metabolism. It is related to lysosomes and can be induced by exogenous substances (Chen et al., 2007; Chi et al., 2017). AKP is the most important metabolic enzyme and plays a crucial role in the absorption and utilization of nutrients in aquatic animals, enhancing the immunity of aquatic animals (Liu et al., 2012; Pinoni and López Mañanes, 2004). PO is an important enzyme in the prophenoloxidase (proPO) system, a key systemic enzyme for recognizing non-self substances in invertebrates. It is closely related to the immunity of the crustaceans (Sritunyalucksana and Söderhäll, 2000). In this study, dietary ELE increased the activities of ACP, AKP and PO of crayfish. A previous study observed that the administration of Mojave yucca (Yucca schidigera) extract enhanced ACP, AKP and PO activities in whiteleg shrimp (Litopenaeus vannamei) (Yang et al., 2015). Many plant extracts can improve the non-specific immunity of aquatic animals (Cheng, 2019; Tan et al., 2017; Wu et al., 2010). Yang et al. reported that dietary ELE significantly improved muscle antioxidant capacity and flesh quality of grass carp (Yang et al., 2021). No studies about the effects of dietary ELE on the non-specific immunity in crayfish have been reported. Further studies are needed to determine if the mechanisms underlying ELE enhanced non-specific immunity are comparable to those found in other terrestrial animals.
Antimicrobial peptides (AMPs), such as HEM, lysozymes (LZM) and ALF, play important roles in the innate immunity of aquatic animals (Destoumieux-Garzon et al., 2001; Ren et al., 2012). The regulation of immune genes is closely related to the regulation of immunity in aquatic animals (Tan et al., 2018). Previous studies reported that E. ulmoides could promote immunity by regulating the secretion of proinflammatory cytokines (Kim et al., 2009; Kim et al., 2012). Similarly, the high expression of AMPs implies an increase in disease resistance in aquatic animals (Shi et al., 2014; Shimizu et al., 2003; Sruthy et al., 2017). In the present study, the results suggested that diet supplement ELE significantly up-regulated the mRNA levels of C-LZM and ALF in the hepatopancreas of crayfish compared with the control group. However, the regulation of AMP gene expression by dietary ELE at the level of mRNA translation was not reported previously in aquatic animals. We demonstrated that ELE induced the expression of the AMP gene. Thus, more research needs to be done to clarify the molecular mechanism.
Plants or plant extracts can improve the resistance of aquatic animals to environmental stress such as low temperature, toxins, heavy metals (Da Silva et al., 2021; Farag et al., 2021; Liu et al., 2012). In the present study, supplementation of ELE to diets significantly improved the resistance of red claw crayfish against MC-LR stress. The increased SR of crayfish to environmental stress, as described for other aquaculture species, can result in increased health status and positive immune response of crayfish (Wang et al., 2015). However, few studies have reported the effects of plant extracts on immune-related genes in crayfish under MC-LR stress. Therefore, we analyzed the immune status of crayfish in each feed group under MC-LR stress. In the current study, supplementation of ELE to diets significantly up-regulated the mRNA levels of SOD, GPx, Se-GPx, GST1, ALF and HEM in the hepatopancreas of crayfish under MC-LR stress compared with the control group. The antioxidant system of crayfish neutralizes the damaging effects of ROS, and the antioxidant system with GSH plays a vital role in MC-LR depuration in crayfish (Yuan et al., 2016). In MC-LR stress experiments, the expression levels of SOD, GPx, Se-GPx and GST1 in crayfish were significantly increased in Diet 3 and Diet 4 groups. After a challenge with MC-LR, crayfish survival was significantly higher in Diet 3 and Diet 4 groups than in the control group, which may be due to the increased effects of ELE on the immune system of crayfish (Raissy et al., 2022). These results are similar to those reported previously (Xie et al., 2022; Yuan et al., 2015). Plant extracts can improve the survival of aquatic animals in stressful environments by enhancing immunity (Liu et al., 2020; Tan et al., 2017; Wang et al., 2011; Wang et al., 2015). Xie et al. reported that Tian-Dong-Tang-Gan powder could improve the resistance of whiteleg shrimp by inducing the expression levels of GPx, Mn-SOD and ACP in the hepatopancreas of the nitrite exposure (Xie et al., 2022). Changes in SOD, GPx, GST, ALF, HEM and LZM gene expression levels suggested that MC-LR induced ROS production and crayfish made the corresponding reaction by regulating these immune-related factors in vivo (Yuan et al., 2016; Yuan et al., 2015). These results indicated that ELE could increase or maintain the mRNA expression levels of SOD, GPx, Se-GPx, GST1, ALF, HEM and C-LZM in the hepatopancreas of juvenile red claw crayfish to regulate the ability of crayfish and resist MC-LR stress, reducing the damage of hepatopancreas caused by MC-LR in juvenile red claw crayfish.