A simple in vitro method to evaluate the toxicity of functional additives used in shrimp aquaculture

Graphical abstract


G R A P H I C A L A B S T R A C T A B S T R A C T
To mitigate the economic losses provoked by disease outbreaks, shrimp producers employ therapeutic additives. However, important issues such as the toxicity of these products on shrimp are not always considered. In vivo toxicity assays require a lot of time and large economic and physical resources. Here, we describe an in vitro procedure to evaluate the toxicity of functional additives, used in the production of shrimp Penaeus vannamei. This method adapted the cell viability assay based on the reduction of tetrazolium salts (MTT) to primary cell cultures of shrimp hemocytes.
A simple and reliable tool that requires few physical and economic resources to evaluate in short time (6 h) the cytotoxic effect of therapeutic products and additives to be included in shrimp culture This inexpensive method requires only a modified Hank's balanced salt solution (HBSS) containing Ca 2+ and Mg 2+ to keep hemocytes metabolically active to successfully carry out the cytotoxicity assay This toxicity in vitro assay does not require exposure of the shrimp to compounds at toxic concentrations.

Background
The expansive growth of the shrimp aquaculture industry is accompanied by the disease outbreaks. To mitigate the economic losses, shrimp producers employ therapeutic additives, such as antibiotics, immune modulators, organic acids, essential oil and antioxidants. However, important issues such as the toxicity of these products on shrimp are not always considered. While in vivo toxicity assays require considerable time and economic resources [1], the development of easy and robust in vitro protocols is highly relevant. The cell viability assay developed by Mosman [2], based on the reduction of tetrazolium salts (3-(4,5-dimethylthiazol-2-yl) À2,5-diphenyltetrazolium bromide) (MTT), is widely used to measure in vitro cytotoxic in eukaryotic cells [2,3]. To develop an in vitro toxicity test suited for the assessment of the toxicity of feed additives for shrimps, we adapted this Mosman protocol to primary cell cultures of shrimp hemocytes. This fast and inexpensive assay can be used by the shrimp industry to determine non-toxic therapeutic doses of functional additives as a preapplication process in in vivo trials, and shrimp farms.

Hemograma
1 Fix an aliquot of the hemolymph using formaldehyde 3.7%, in a V/V ratio.
2 Count the hemocytes using 10 mL of the fixed hemolymph with a hematocytometer (Neubauer chamber). Adjust hemocytes concentration to 1 Â10 7 cells ml À1 , using sodium citrate at 5% in sodium chloride solution 2%. 2 After incubation, add 10 mL of MTT (5 mg/ml MTT in milliq water) to all wells and incubate for 120 min at 25 C. Keep in the dark. 3 After 120 min of incubation, shrimp hemocytes become stained with formazan and the wells turn purple (Fig. 1B). Remove the supernatant, and add 150 mL of isopropanol containing 0.04 N HCl.
Homogenize vigorously to dissolve the formazan crystals (Fig. 1A), placing the microplate onto an ice bed. 4 Read at 620 nm in a microplate reader. The percentage of cell viability is obtained using the formula.

Additional information
The toxicity assay developed by Mossman [2] has been applied in several studies with eukaryotic cells and it is widely used to measure cytotoxic, and antiproliferative activity of compounds [3]. This test has also been used by Jose et al. [4] to estimate the viability of hemocytes of Penaeus monodon shrimp to study the White Spot Syndrome Virus in vitro. In our study we used the procedure described by Muñoz et al. [5] to perform primary cell cultures of P. vannamei haemocytes. This method requires only a solution of MHBSS containing Ca 2+ and Mg 2+ to keep hemocytes metabolically active (Fig. 1B) to successfully carry out the cytotoxicity assay.
We used this assay to evaluate the toxic doses of different aquaculture additives, such as essential oils and antibiotics amongst others. In the (Fig. 2), we illustrated the effect of essential oil of Origanum vulgare (18% oil of O. vulgare), over shrimp hemocyte viability. O. vulgare essential oil is rich in thymol and carvacrol, phenolic compounds with several bioactivities [6], antioxidant [7][8][9], microbicidal [10-  12] and immune modulator [13]. The results obtained with our in vitro test indicated that O. vulgare essential oil is not toxic for hemocyte at concentration 0.1, 1 and 10 ppm as no significant differences in cell viability were found between control and treatments. At these concentrations less than 10% of the hemocytes were affected (Fig. 2). Based on these results we performed an in vivo study. Post-larvae from P. vannamei shrimp in PL-1, were exposed to O. vulgare essential oil, using several doses between 0.1 and 10 ppm. The post-larvae survival was slightly affected 10% only at 10 ppm. These results were consistent with in vitro results, indicating the effectiveness of in vitro assay to determine no toxic therapeutic doses of functional additives as a pre-application process in in vivo trials. Also, we evaluated the toxicity in vitro of oxitetracicline (OTC) and florfenicol, antibiotics commonly used in aquaculture. Recording toxicity for oxytetracycline and florfenicol in shrimp hemocytes at concentrations of 4000 and 2000 mg/ml respectively. At these concentrations 15% of the cell viability was affected. Combining this information together with data of bacterial resistance, researchers and producers could effective doses of these drugs at commercial scale.
In conclusion, this test is a simple and reliable tool that requires few physical and economic resources to evaluate in short time (6 h) the cytotoxic effect of therapeutic products and additive to be included in shrimp culture.