Effect of Solanum procumbens Lour. Extract on Survival, Growth Performances, Immune Responses and against Vibrio parahaemolyticus causing Acute Hepatopancreas Necrosis Disease in White Leg Shrimp (Litopenaeus vannamei)

The experiment was conducted from March 2021 to September 2021 at Tra Vinh University, Tra Vinh province, Vietnam. Dried S. procumbens L. was bought from a local traditional medicine store. The extraction of S. procumbens L. was applied using the protocol described by Chaweepack et al., (2015). Briefly, the dried S. procumbens L. was ground into fine powder. After that, the powder was added to a 70% alcohol solution at a ratio of 1:10 and left for 24 h before being filtered with paper. The collected liquid was evaporated at 50°C using a vacuum evaporation machine. The remaining crude extract was freeze-dried and stored at 4°C for further experiments. Shrimp at postlarvea 12 (specific pathogen free-SPF) was reared to reach required size for the experiment. Shrimp at 5.33±0.13 g was selected to place in 0.5 m³  tanks containing 15ppt aerated seawater. The density of shrimp was 50 individuals per tank. The shrimp was fed commercial pellets supplemented with four different ratios of S. procumbens L. extract at 0.0%, 0.5%, 1.0% and 1.5% four times per day. Each treatment was three time replication. Before feeding, S. procumbens L. extract was weighted and dissolved in sterilized water before being sprayed on the pellets. After that, the pellets were coated with squid oil at 5ml per kg of feed. Water parameters such as salinity, pH, oxygen and temperature in each tank were measured daily; mean while, ammonia and hydrosulfic were measured every 3 days. Salinity was measured by master refractometer (Atago, Japan), pH and temperature by pH/Cond/Temp Pocket Meter (EZDO 7200, Taiwan), dissolved oxygen by oxygen meter (SevenGo pro; Mettler Toledo, USA), ammonia and hydrogen sulfide by sera test kit (Germany). During the experiment, environmental parameters were maintained at suitable levels for shrimp growth: dissolved oxygen was above 4 ppm, pH between 7.5-8, temperature between 28-29°C, salinity at 15 ppt and ammonia and hydrogen sulfide were undetectable. Siphoning and water exchange was carried out at 10% every 3 days. At the end of experiment, survival rate (SR), growth responses and immune parameters of experimental shrimp were examined. SR and growth responses including weight gain (WG), absolute growth rate (AGR) and specific growth rate (SGR) of experimental shrimp was calculated following formulas described by Chandran et al., (2016). In immune response examination, hemolymph collection for total haemocyte count (THC) and phenoloxidase (PO) activity measurement followed protocols described by Hernández López et al., (1996). THC was determined following the protocol described by Kakoolaki et al., (2011). The PO activity was determined in duplicate following the protocol described by Hernández López et al., (1996) and Widanarni et al., (2020).
 
To observe the effects of the treatment against Vp_AHPND, pirAB gene positive V. parahaemolyticus was previously isolated from AHPND shrimp stored at -80°C at the Laboratory of Aquaculture Disease Management, Tra Vinh University and it was recovered on trypsin soybean agar (TSA) (Himedia, India). The pirAB genes of V. parahaemolyticus isolate were rechecked by duplex PCR method using the protocol described by Han et al., (2015). Shrimp fed diets supplemented 0.0%, 0.5%, 1.0% and 1.5% after 30 days was subjected to challenge tests with three time replications. The challenge test followed the protocol described by Loc et al., (2013) and Hong To et al., (2020). Ten shrimps were placed in each plastic container containing 20L of 15 ppt seawater. V. parahaemolyticus harboring pirAB genes recovered on TSA was diluted in sterilized saline water and added into experiment containers. The density of AHPND V. parahaemolyticus in plastic containers was adjusted at 10⁶ CFU/ml. The experiment was observed over two weeks for the cumulative mortality and clinical signs of infected shrimp. Moribund shrimp was picked-up for re-isolation of Vp_AHPND. The collected data was analyzed for normal distribution and variance homogeneity. One-way ANOVA was applied and Turkey HSD was employed for comparison. Significant differences were considered at P<0.05. SPSS software (version 20. IBM,USA) was used for statistical analysis.

Survival, growth performance and immune responses
 
Fig 1 indicates that shrimp fed control diet and S. procumbens L. extract supplementation showed no significant difference (P>0.05) in survival rates (76%-78%) of shrimp among treatments after the period of 30 days. There were also no significant differences (P>0.05) in growth responses between shrimp fed with and without extract supplementation (Table 1). Regarding immune responses, although THC and PO activity of animals fed 1.0% was higher than that of animals fed 0.0% and 0.5% extract, they were no significant differences (P>0.05). Notably, THC (Fig 2) and PO activity (Fig 3) of animals fed 1.5% extract were significantly higher than those recorded in other treatments (P<0.05).
 

 

 

 

 
Many medicine plants could be accepted as antibiotic alternative solution in aquaculture because they contain activate molecules that could benefit for growth and immune system of animals (Pu et al., 2017; Kaur et al., 2022; Bharathi et al., 2021; Jha et al., 2022). Previous studies demonstrated the effectiveness of herbal extract or herbal products on survival and immune system of shrimp (Chandran et al., 2016). In this study, shrimp fed S. procumbens L. extract displayed an improvement in immune parameters such as THC and PO value. Similarly, Chandran et al., (2016) reported that Penaeus monodon postlarvae fed polyherbal formulation (aqualmmu) showed better performances of immune parameters such as THC, superoxide anion activity, PO activity, lysozyme activity, plasma protein content and bactericidal activity in comparison to shrimp without herbal supplementation. Recent study by Xie et al., (2021) also indicated that white leg shrimp fed diets supplemented Tian-Dong-Tang-Gan powder increased the activities of PO, acid phosphatase, superoxide dismutase and alkaline phosphatase in the shrimp hemolymph during the 28-day feeding trials. As reported by AftabUddin et al., (2021), the administration of Padina tetrastromatica extract improved the immune capacity of P. monodon such as increasing THC, PO activity and superoxide anion concentration. Other research stated that a combination of enrofloxacin and Chinese herbal product (San-Huang-San) can enhance immune parameters of white leg shrimp including THC and PO activity (Zhai and Li, 2019).
 
Against Vp_AHPND 
 
Table 2 indicates that after 10 days challenged with Vp­_AHPND, the accumulate mortality of shrimp fed S. procumbens L. extract at 1.0% and 1.5% (52.4%) was significantly lower (P<0.05) than that observed on animals fed diet supplemented S. procumbens L. extract at 0.0% and 0.5% (100% and 90%, respectively). The infected shrimp showed the clinical symptom of AHPND such as pale and atrophied hepatopancreas, empty stomach and midgut. V. parahaemolyticus harboring pirAB genes was also isolated from moribund shrimp in the challenge experiment.
 

       
It is known that V. parahaemolyticus is the causative agent of AHPND in shrimp and has caused large economic losses for shrimp farming worldwide (Loc et al., 2013). In this study, S. procumbens L. extract was shown to reduce the mortality of Vp_AHPND challenged shrimp. Other research also noted that plant extracts can protect shrimp from AHPND infection. Jha et al., (2016) stated that white leg shrimp fed a mixer of natural essential oils extracted from various herb species Lavandula latifolia, Pinus sylvestris, Jasminum officinale, Citrus limon, Prunus avium, Viola odorata, Gardenia jasminoides, Cocos nucifera, Rosa damascene and Eucalyptus globulus showed no mortality as challenged with Vp_AHPND. Babikian et al., (2019) also indicated the Pondguard (Registration no. D 16060285-HBC) consisting of natural oils, lavender oil, eucalyptus oil and pine oil can help to improve survival rate by approximately 50% of shrimp from Vp_AHPND challenge. According to Gamboa-Barraza et al., (2021), white leg shrimp fed macroalgae Gracilaria vermiculophylla and Ulva flexuosa extract provided a survival rate at 60%-67% which was twice the amount of survival in the positive control at 24 h post-infection with Vp_AHPND.

In conclusion, although S. procumbens L. extract did not enhance survival rate and growth performances of white leg shrimp in this study, the extract can improve innate immune parameters such as increasing THC and PO value and reducing the mortality of shrimp challenged with VpAHPND. Therefore, S. procumbens L. could be a potential material for enhancing innate immunity of white leg shrimp and protecting shrimp from AHPND infection.

This study was funded by the Tra Vinh University as in contract number 204/2020/HÐ.HÐKH-ÐHTV. We express our deep thanks to Ms. Nguyen Thi Hong Muoi, Ms. Lam Thi Yen Trinh, Ms. Tran Thi Phuong Lan and Mr. Thach Anh Tuan for their great support of this study.

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