Transcriptomic Analysis of Gill and Kidney from Asian Seabass (Lates calcarifer) Acclimated to Different Salinities Reveals Pathways Involved with Euryhalinity

Abstract

Asian seabass (or commonly known as barramundi), Lates calcarifer, is a bony euryhaline teleost from the Family Latidae, inhabiting nearshore, estuarine, and marine connected freshwaters throughout the tropical Indo-West Pacific region. The species is catadromous, whereby adults spawn in salinities between 28 and 34 ppt at the mouth of estuaries, with resultant juveniles usually moving into brackish and freshwater systems to mature, before returning to the sea to spawn again as adults. The species lives in both marine and freshwater habitats and can move quickly between the two; thus, the species' ability to tolerate changes in salinity makes it a good candidate for studying the salinity acclimation response in teleosts. In this study, the transcriptome of two major osmoregulatory organs (gills and kidneys) of young juvenile Asian seabass reared in freshwater and seawater were compared. The euryhaline nature of Asian seabass was found to be highly pliable and the moldability of the trait was further confirmed by histological analyses of gills and kidneys. Differences in major expression pathways were observed, with differentially expressed genes including those related to osmoregulation, tissue/organ morphogenesis, and cell volume regulation as central to the osmo-adaptive response. Additionally, genes coding for mucins were upregulated specifically under saline conditions, whereas several genes important for growth and development, as well as circadian entrainment were specifically enriched in fish reared in freshwater. Routing of the circadian rhythm mediated by salinity changes could be the initial step in salinity acclimation and possibly migration in euryhaline fish species such as the Asian seabass.

Keywords: Asian seabass; acclimation; euryhalinity; fish; freshwater transcriptome; marine transcriptome; osmoregulation; pliable trait; transcriptome.

Figure 1

Flow chart outlining the experimental design for assessing the Asian seabass transcriptome under different salinity conditions. Abbreviations: dph: days post hatch; gills (SG1) and kidneys (SK1) from seawater grown fishes at 65 dph; gills (FG1) and kidneys (FK1) from freshwater grown fishes at 65 dph; gills (SG2) and kidneys (SK2) from seawater grown fishes at 86 dph; gills (RSG2) and kidneys (RSK2) from fishes returned to seawater at 86 dph.

Figure 2

Histological analysis of Asian seabass gills showed a highly plastic phenotype with fishes transferred to freshwater showing a distinct decrease in chloride cell numbers which was regained upon being transferred back to seawater. Transverse section of the gills of Asian seabass (A) SG1, (B) FG1, (C) SG2, (D) RSG2. The sections were stained with Von Kossa staining. The scale bar is 20 µm. A multiple pairwise comparison test of chloride cell numbers between the four different groups showed that each group was significantly different (p < 0.05) from the other (E). Abbreviations: PL: primary lamella; SL: secondary lamella; CC: chloride cells; CVS: central venous sinus; MC: mucus cells; gills (SG1) from sea water grown fishes at 65 dph; gills (FG1) from fresh water grown fishes at 65 dph; gills (SG2) from sea water grown fishes at 86 dph; gills (RSG2) from fishes returned to seawater at 86 dph.

Figure 3

Histological analysis of Asian seabass grown in seawater shows structural changes in the Bowman’s capsule, glomerulus, and papillary ducts of the kidney upon being transferred and grown in freshwater. Transverse section of the Asian seabass kidney (A) SK1, (B) FK1, (C) SK2, (D) RSK2. The sections were stained with H&E. The scale bar is 20 µm. Multiple pairwise comparison test of glomerular size between the four different groups (E). The letters above each bar (a: SK1; b: FK1; c: SK2; d: RSK2) indicates the groups from which it is significantly different (p < 0.05). Abbreviations: BS: Bowman’s space; G: glomerulus; DT: distal tubules; kidneys (SK1) from seawater grown fishes at 65 dph; kidneys (FK1) from freshwater grown fishes at 65 dph; kidneys (SK2) from seawater grown fishes at 86 dph; kidneys (RSK2) from fishes returned to seawater at 86 dph.

Figure 4

Summary of differentially expressed genes in the gills and kidneys of Asian seabass acclimated to different salinity conditions. (A) The graph shows the number of differentially expressed genes (up- and downregulated) under different conditions—FG1 vs. SG1, FK1 vs. SK1, RSG2 vs. SG2, and RSG2 vs. FG1. (B,C) Venn diagrams showing the unique differentially expressed genes as well as genes shared between the different salinity conditions or organs. Abbreviations: Refer to Figure 1.

Figure 5

The euryhaline trait is pliable and involves the differential expression of a core set of genes. The top 25 differentially expressed genes in the FG1 vs. SG1 comparison (blue diamonds; 1–25) were checked for in RSG2 comparison vs. SG2 (orange circles; 1–25). The majority of these transcripts were still upregulated in RSG2 compared to SG2 and all of them were expressed to a much lesser extent compared to the FG1 vs. SG1. Thus, the top 25 upregulated genes (FG1 vs. SG1) seem to be reinstating themselves to the saline conditions in the RSG2 group of fishes. Abbreviations: Refer to Figure 1.

Figure 6

Gene ontology analyses of (A) Biological processes, (B) molecular Functions, and (C) Cellular components using the Web Gene Ontology Annotation Plot (WEGO) of the Asian seabass gill and kidney transcriptomes under different salinity conditions. Only categories with 15 or more protein associations are shown. Blue: upregulated in FG1 vs. SG1; Yellow: downregulated in FG1 vs. SG1; Purple: upregulated in FK1 vs. SK1; Green: downregulated in FK1 vs. SK1. Abbreviations: Refer to Figure 1.

Figure 7

Pathways significantly (p < 0.05) enriched amongst the differentially expressed transcripts in the osmoregulatory organs. The pathway is indicated on the Y-axis and the proportion of genes in the pathway is indicated on the X-axis. Statistical significance was determined as Benjamini–Hochberg adjusted p-value < 0.05. Abbreviations: M: Metabolism; OS: Organismal system; EIP: Environmental information processing; HD: Human disease; CP: Cellular processes. Additional information can be found in Supplementary Table S5.