Na+/H+ exchanger (NHE) in Pacific white shrimp (Litopenaeus vannamei): Molecular cloning, transcriptional response to acidity stress, and physiological roles in pH homeostasis

Abstract

Na+/H+ exchangers are the most common membrane proteins involved in the regulation of intracellular pH that concurrently transport Na+ into the cells and H+ out of the cells. In this study, the full-length cDNA of the Na+/H+ exchanger (NHE) from the Pacific white shrimp (Litopenaeus vannamei) was cloned. The LvNHE cDNA is 3167 bp long, contains a 5'-untranslated region (UTR) of 74 bp and a 3'-UTR of 456 bp and an open reading frame (ORF) of 2637 bp, coding for a protein of 878 amino acids with 11 putative transmembrane domains and a long cytoplasmic tail. LvNHE shows high sequence homology with mud crab NHE at the amino acid level. LvNHE mRNA was detected in the hepatopancreas, gill, eyestalk, skin, heart, intestine, muscle, brain and stomach, with the highest abundance in the intestine. In the shrimp intestinal fragment cultures exposed to gradually declining pH medium (from pH 8.0 to pH 6.4), the LvNHE mRNA expression was significantly stimulated, with the highest response when incubated in pH 7.0 medium for 6 h. To investigate the functional roles of LvNHE in pH regulation at the physiological and cellular levels, the LvNHE mRNA expression was silenced by siRNA knockdown. Upon low-pH challenge, the hemolymph pH was significantly reduced in the LvNHE mRNA knockdown shrimp. In addition, knockdown of LvNHE mRNA reduced the recovery capacity of intracellular pH in intestinal fragment cultures after acidification. Altogether, this study demonstrates the role of NHE in shrimp response to low pH stress and provides new insights into the acid/base homeostasis mechanisms of crustaceans.

Fig 1. The full-length cDNA sequence and its deduced amino acid sequence of LvNHE.

The start codon (ATG) and stop codon (TGA) are shown in red. The transmembrane (TM) domains are indicated. The NEXCaM regulatory region is boxed. The consensus polyadenylation (AATAAA) signal is underlined.

Fig 2. Structural domains of LvNHE and phylogenetic analysis.

(A) Structural domains of LvNHE predicted by using the SMART program. The 11 transmembrane (TM) domains and the NEXCaM_BD feature with respective locations are indicated. (B) Phylogenetic analysis of NHEs in various species by using the Neighbor-Joining method with a bootstrap value of 1000. The newly identified LvNHE is marked by a triangle.

Fig 3. Tissue expression pattern and pH-induced expression of LvNHE.

(A) Expression profiles of LvNHE mRNA in various L. vannamei tissues, including the hepatopancreas (Hp), gill (Gi), eyestalk (Es), skin (Sk), heart (Ht), intestine (In), muscle (Ms), brain (Br) and stomach (St). (B) LvNHE expression levels in the intestines after low pH challenge. The LvNHE mRNA levels were normalized by β-actin expression. The data are expressed as the mean±SE (n = 3), and experimental groups denoted by the same letter represent a similar level (P>0.05, ANOVA followed by Fisher’s LSD test).

Fig 4. Effect of LvNHE mRNA knockdown on the hemolymph pH upon low pH challenge.

(A) Expression of LvNHE transcripts in shrimp intestines after injection of DEPC-H₂O, NC-siRNA and LvNHE siRNA-1, -2 and -3 at 6 and 12 h. (B) The hemolymph pH of LvNHE-siRNA-injected shrimp upon low (5.8) pH challenge for 6 and 12 h. In this case, the dosages for all siRNA groups were 1 μg/g bwt, and the individual numbers for all groups were 3. For mRNA and hemolymph pH measurements, the data are expressed as the mean±SE, and experimental groups denoted by the same letter represent a similar level (P>0.05, ANOVA followed by Fisher’s LSD test).

Fig 5. Effect of LvNHE mRNA knockdown on pH_i recovery in intestinal fragment culture.

(A) The calibration curve for transforming the fluorescence ratio (490/440) into a pH value. (B) pH_i recovery in intestinal fragment culture following intracellular acidification upon readdition of Na⁺. Intracellular pH was measured, and an acid load was applied using the NH₄Cl-prepulse technique in the absence of extracellular Na⁺. (C) The pHi recovery rate (dpH/dt) of intestinal fragment culture from shrimp injected with LvNHE siRNA-2 for 6 and 12 h. The data for calibration curve and pHi recovery rate are expressed as the mean±SE (n = 8), and experimental groups denoted by the same letter represent a similar level (P>0.05, ANOVA followed by Fisher’s LSD test).