Novel spikey ionocytes are regulated by cortisol in the skin of an amphibious fish

Abstract

Cortisol is a major osmoregulatory hormone in fishes. Cortisol acts upon the gills, the primary site of ionoregulation, through modifications to specialized ion-transporting cells called ionocytes. We tested the hypothesis that cortisol also acts as a major regulator of skin ionocyte remodelling in the amphibious mangrove rivulus (Kryptolebias marmoratus) when gill function ceases during the water-to-land transition. When out of water, K. marmoratus demonstrated a robust cortisol response, which was linked with the remodelling of skin ionocytes to increase cell cross-sectional area and Na⁺-K⁺-ATPase (NKA) content, but not when cortisol synthesis was chemically inhibited by metyrapone. Additionally, we discovered a novel morphology of skin-specific ionocyte that are spikey with multiple cell processes. Spikey ionocytes increased in density, cell cross-sectional area and NKA content during air exposure, but not in metyrapone-treated fish. Our findings demonstrate that skin ionocyte remodelling during the water-to-land transition in amphibious fish is regulated by cortisol, the same hormone that regulates gill ionocyte remodelling in salinity-challenged teleosts, suggesting conserved hormonal function across diverse environmental disturbances and organs in fishes.

Keywords: Kryptolebias marmoratus; fish; gill; ionoregulation; ions; metyrapone.

Figure 1.

Metyrapone treatment inhibits an increase in whole-body cortisol levels (ng g⁻¹ tissue) in air-exposed adult mangrove rivulus (Kryptolebias marmoratus). Different letters represent statistical significance (p < 0.05). Mean ± s.e.m. (air: n = 7–9; metyrapone: n = 3–4). (Online version in colour.)

Figure 2.

Comparison of round and spikey ionocyte morphologies in the skin of K. marmoratus. (a,b) Representative X-Z optical sections reconstruction of NKA-labelled ventral skin ionocytes (red) against a DAPI stained (blue) background. Insets show X-Y maximum intensity projection images of the same cells; (a) depicts a typical round morphology ionocyte (shape factor = 0.95), and (b) a spikey ionocyte morphology (shape factor = 0.26). (c) Shape factor comparison of all round and spikey ionocyte morphologies across all treatments. Asterisks indicate a significant difference (unpaired t-test: p < 0.0001) between cell types. (d) Ionocyte cross-sectional area comparison of all round and spikey cells across treatments. Asterisks indicate a significant difference as above. (e) Total cell fluorescence comparison of all round and spikey skin ionocytes. All error bars denote mean ± s.e.m. (n = 22 per cell type). (Online version in colour.)

Figure 3.

Air exposure results in skin ionocyte remodelling when K. marmoratus are able to mount a cortisol response. (a) Mean density of round and spikey ionocytes (cells mm⁻²) across treatment groups. The X in the legend indicates a significant interaction (two-way ANOVA; interaction, p < 0.0001). Data were therefore split by cell type and analysed separately. Different uppercase letters indicate significant differences between treatments in the round cells, and different lower-case letters indicate significant differences between treatments in the spikey cells. The inset graph shows the overall (combined spikey and round) ionocyte density across treatments (one-way ANOVA: p < 0.05). (b) Ionocyte cell cross-sectional area (μm²) of round and spikey ionocyte morphologies across treatment groups (two-way ANOVA: treatment, p < 0.0001, cell type, p < 0.0001). Dissimilar letters indicate significant differences between treatments and the dagger in the legend indicates significant differences within each treatment between round and spikey cells. The inset graph shows the change in overall ionocyte cross-sectional area across treatments (one-way ANOVA: p < 0.05). (c) Cell fluorescence of round and spikey skin ionocytes across treatment groups (two-way ANOVA: treatment, p < 0.05, cell type, p < 0.05). Letters and dagger indicate significances as in (b) above. The inset graph shows the overall cell fluorescence across treatments (one-way ANOVA: p < 0.05). Each point represents an individual fish (n = 6–9 per treatment) and error bars denote the mean ± s.e.m. (Online version in colour.)