The establishment of shrimp environmental stressors

Description

Commercially cultivated shrimps are susceptible to the negative effects of stresses caused by unfavorable environmental conditions and pathogenic infections. These stresses can produce a range of outcomes from slow growth to high mortality, and all of these result in economic losses. The shrimp aquaculture industry would therefore benefit greatly from a diagnostic platform that could used to monitor cultured shrimp populations and provide early warning of stressful conditions. To do this, we first set up different stressful conditions (e.g. hypoxia, changes in salinity or pH, pathogen infection) for shrimps, and samples are collected from each treatment for further assays. Next, we use microarrays to identify shrimp genes that are up-regulated at the transcriptional level by various stressful conditions. Candidate stress marker genes will then be further validated and characterized using real-time PCR.

Brief protocol

Establishment of stressful conditions

Experimental animals

The Litopenaeus vannamei (average weight 10 g) used in the experiments were reared in farm at the NTOU Aquatic Animal Center. These shrimps were transported to our laboratory and acclimated in constantly aerated tanks (8-10 shrimps per tank) containing artificial sea water (28 ppt salinity, pH 8.0, 25℃) at least 5 days before experimentation. They were fed commercial shrimp pellets twice daily. Shrimps were not fed for 24h before the experiment, and seawater renewal was stopped during the experiment. At least three replicate tanks were used for each treatment.

Hypoxia stress

The required low dissolved oxygen (DO) level (1.5 mg l^-1) of the experimental groups was established by bubbling nitrogen gas and air through the seawater. Oxygen levels were monitored by a DO meter that was checked every 2 h. The saturated DO level (6.5 mg l^-1) of control groups was maintained by continuous aeration. A single shrimp was randomly selected from each replicate tank at 0, 12, 24, 48, 72 h, and tissue samples (gills, stomach, lymphoid organ, pleopod) were collected and stored at -80℃ for further study. No shrimps died during the experiment.

Salinity stress

The desired low salinity (15 ppt), normal salinity (28 ppm)) and high salinity (40 ppt) were obtained by adding sea salts to fresh water. Salinity was checked and adjusted with a salinity meter. A single shrimp was randomly selected from each replicate tank at 0, 12, 24, 48, 72 h, and tissue samples (gills, stomach, lymphoid organ, pleopod) were collected and stored at -80℃ for further study. No shrimps died during the experiment.

pH stress

The pH level of artificial seawater (28 ppm salinity) was adjusted by adding 4N NaOH or 4N HCl to obtain high pH 9.3 or low pH 5.6, respectively. pH was continuously monitored by a pH meter, and checked every 2 h. The control group was kept in seawater with pH 8.0. A single shrimp was randomly selected from each replicate tank at 0, 12, 24, 48, 72 h, and tissue samples (gills, stomach, lymphoid organ, pleopod) were collected and stored at -80℃ for further study. No shrimps died during the experiment.