Expression of aqp genes and AQP transporter proteins in juvenile stages of stinging catfish
The relative expressions of mRNAs for different aqp genes (Fig. 2) and their translated AQP transporter proteins (Fig. 3) were analyzed both in the anterior and posterior parts in three juvenile stages (S1, 7-days old; S2, 14-days old; S3, 21-days old, Fig. 1) of stinging catfish. The relative changes in the levels of expression of mRNAs of different aqp genes and their translated proteins in the S2 and S3 stages were compared with the levels found in the S1 stage.
In general, the levels of mRNA for different aqp genes were found to be more expressed in S2 and S3 juveniles compared to the levels found in S1 juveniles in both the anterior and posterior parts of juveniles (Fig. 2a & b). In general, the levels of mRNAs for aqp1, aqp1b, aqp3, aqp4, aqp7, aqp8, aqp11, and aqp12 were found to be significantly higher in the anterior regions of S2 and S3 juveniles than in S1 juveniles.
As shown in Fig. 2a, the expression of mRNA for aqp1 gene was found to be 8.1-fold higher in S2 juveniles and 5.5-fold higher in S3 juveniles compared to the level found in S1 juveniles. The expression of mRNA for aqp1b gene was also found to be 31.1- and 23.0-fold higher in S2 and S3 juveniles, respectively, compared to the level found in the S1 juvenile. Whereas, in cases of aqp3, aqp4, and aqp7 genes, the mRNA levels were found to increase significantly by 2.5-, 3.0- and 2.7-fold in S2 juvenile, respectively, and by 3.1-, 3.3- and 3.5-fold in S3 juvenile, respectively, compared to the levels found in S1 juvenile. In cases of aqp8, and aqp11, the fold increases of mRNA levels were again found to be much higher in S2 and S3 juveniles compared to levels seen in S1 juveniles, which were 24.5-, and 11.3-fold higher in S2 juveniles, and 37.3- and 27.5-fold higher, respectively, in S3 juvenile. In the case of aqp12 gene, the level of mRNA expression was again higher in S2 juveniles by 6.5-fold, and by 5.4-fold in S3 juveniles compared to the level found in S1 juveniles.
Similarly, the posterior region of three juvenile stages of stinging catfish showed some variations in the expression of mRNA transcripts for different aqp genes (Fig. 2b). For aqp1, there were 7.7- and 17.4-fold more expression in S2 and S3 juveniles, respectively, compared to the level found in S1 juveniles. For aqp1b, the mRNA level was found to be 53.5- and 38.5-fold higher in S2 and S3 juveniles, respectively, compared to the level found in S1 juveniles. Whereas, in the case of aqp3 and aqp4 genes, the expression of mRNAs increased by 6.5-fold and 5.5-fold in S2 juveniles and by 7.2- and 6.3-fold in S3 juveniles compared to the level found in S1 juvenile. In contrast to the anterior part of juveniles, the expression of aqp7 mRNA was found to be 51.0- and 54.7-fold higher in S2 and S3 juveniles, respectively, compared to the level found in S1 juveniles. In cases of aqp8 and aqp12 genes, again, not much of variations in the expression of mRNAs were seen among different juvenile stages. Whereas, in the case of aqp11, the mRNA level was found to be 13.4- and 19.2-fold higher in S2 and S3 juveniles, respectively, compared to the level found in S1 juveniles.
The pattern of relative expression of different AQP transporter proteins in three different juvenile stages (S1, S2, and S3) of stinging catfish showed almost a similar trend comparable to the relative expression of mRNAs of corresponding aqp genes (Fig. 3a & b). In general, the levels of expression of different AQP proteins were found to be higher in S2 and S2 juveniles compared to the levels found in S1 juveniles of stinging catfish. For AQP1, the level of expression was 4.5- and 3.0-fold higher in the anterior region of S2 and S3 juveniles, respectively, compared to the level found in the anterior region of S1 juvenile, and 3.1- and 3.5-fold higher in the posterior region of S2 and S3 juveniles, respectively, compared to the level found in the posterior region of S1 juvenile. For AQP3, the level of expression was 2.4- and 2.9-fold higher in S2 and S3 juveniles, respectively, compared to the level found in the anterior region of S1 juvenile, and 1.7- and 2.1-fold higher in the posterior region of S2 and S3 juveniles, respectively, compared to the level found in the posterior region of S1 juvenile. Similarly, for AQP4, the level of expression in the anterior region of S1 and S2 juveniles were found to be 2.8- and 3.1-fold higher, respectively, compared to the level found in the anterior region of S1 juveniles, and 2.2- and 2.8-fold higher in the posterior region of S2 and S3 juveniles, respectively, compared to the level found in the posterior region of S1 juvenile. Likewise, in the case of AQP7, the fold increase in its expression was found to be 2.9- and 3.3-fold higher in the anterior region and 3.2- and 3.5-fold higher in the posterior region of S2 and S3 juveniles, respectively, compared to S1 juvenile. Whereas in cases of AQP8 and AQP12 proteins, the significant fold increases were seen in S2 and S3 juveniles only in the anterior region compared to S1 juveniles, but without any significant difference in their expression patterns among three stages in the posterior region of juveniles. However, in the case of AQP11, the levels of expression were 3.1- and 3.6-fold higher in the anterior region and 2.4- and 3.4-fold higher in the posterior region of S2 and S3 juveniles, respectively, compared to the level in S1 juvenile.
Expression of aqp transcripts and related proteins in S2 juvenile of stinging catfish during exposure to hypertonicity
The changes in the expression of aqp transcripts were studied both in the anterior and posterior parts of 14-day old juvenile (S2) stinging catfish while exposed to a hypertonic environment (300 mOsmol/L NaCl) for a period of 48 h (Fig. 4). Wide variations in the expression of different aqp genes were noticed in the anterior part of the S2 juvenile of stinging catfish throughout the period of 48 h exposure to hypertonicity (Fig. 4a). The mRNAs for aqp1, aqp4, aqp8, aqp11, and aqp12 genes were found to be significantly upregulated within 6 h of exposure to hypertonicity, followed by a further increase at later stages of exposure. The mRNAs for aqp1, aqp4, aqp8, aqp11 and aqp12 increased maximally by 43.0-fold after 12 h, 48.2-fold after 24 h, 45.9-fold after 24 h, 11.3-fold after 24 h, 4.9-fold after 12 h of exposure, respectively. Whereas, in the cases of aqp1b and aqp3, the expression levels of mRNAs were found to be downregulated during hypertonic exposure. In the case of aqp7, there was an initial downregulation of mRNA expression at an early stage of treatment, followed by an upregulation by 2.5-fold after 12 h of hypertonic exposure.
In the posterior region of the S2 juvenile, the expression of mRNAs for aqp1 and aqp3 genes were seen to decrease significantly throughout the period of exposure to hypertonicity compared to 0 h control (Fig. 4b). Whereas, in the cases of aqp1b, aqp4, aqp7, aqp8, aqp11, and aqp12, the mRNA levels increased significantly during hypertonic exposure and remained increased throughout the period of exposure with maximum rises of 49.5-fold after 48 h, 36.7-fold after 24 h, 27.8-fold after 24 h, 7.3-fold after 48 h, 11.9-fold after 12 h, and 14.4-fold after 24 h, respectively.
Parallel to the changes in the expression of different mRNA transcripts for aqp genes, the different AQP proteins also showed differential expression patterns in the anterior region of S2 juveniles at different time intervals of hypertonic exposure, as revealed by Western Blot analysis (Figs. 5a & b). In the anterior region, the relative band intensities of AQP1, AQP4, AQP7, AQP8, AQP11 and AQP12 increased significantly with a maximum rise of 7.7-fold at 24 h, 8.1-fold at 24 h, 4.1-fold at 12 h, 7.9-fold at 24 h, 2.9-fold at 24 h and 2.1-fold at 12 h of treatment, respectively. On the other hand, the expression of AQP3 was seen to remain downregulated throughout the period of hypertonic exposure as compared to 0 h control.
Upon exposure to hypertonicity, the relative expression of different AQP proteins in the posterior part of S2 juvenile was seen to increase significantly except for AQP3, which showed a decreasing trend in its expression throughout the period of hypertonic exposure (Figs. 5a & 5b). The AQP1, AQP4, AQP7, AQP8, AQP11, and AQP12 proteins expression increased maximally by 7.9-fold at 48 h, 4.5-fold at 24 h, 4.7-fold at 12 h, 4.2-fold at 48 h, 2.5-fold at 12 h, 3.5-fold at 12 h, respectively.