Effects of Experimental Terrestrialization on the Skin Mucus Proteome of African Lungfish (Protopterus dolloi)

Abstract

Animal mucosal barriers constantly interact with the external environment, and this interaction is markedly different in aquatic and terrestrial environments. Transitioning from water to land was a critical step in vertebrate evolution, but the immune adaptations that mucosal barriers such as the skin underwent during that process are essentially unknown. Vertebrate animals such as the African lungfish have a bimodal life, switching from freshwater to terrestrial habitats when environmental conditions are not favorable. African lungfish skin mucus secretions contribute to the terrestrialization process by forming a cocoon that surrounds and protects the lungfish body. The goal of this study was to characterize the skin mucus immunoproteome of African lungfish, Protopterus dolloi, before and during the induction phase of terrestrialization as well as the immunoproteome of the gill mucus during the terrestrialization induction phase. Using LC-MS/MS, we identified a total of 974 proteins using a lungfish Illumina RNA-seq database, 1,256 proteins from previously published lungfish sequence read archive and 880 proteins using a lungfish 454 RNA-seq database for annotation in the three samples analyzed (free-swimming skin mucus, terrestrialized skin mucus, and terrestrialized gill mucus). The terrestrialized skin mucus proteome was enriched in proteins with known antimicrobial functions such as histones and S100 proteins compared to free-swimming skin mucus. In support, gene ontology analyses showed that the terrestrialized skin mucus proteome has predicted functions in processes such as viral process, defense response to Gram-negative bacterium, and tumor necrosis factor-mediated signaling. Importantly, we observed a switch in immunoglobulin heavy chain secretion upon terrestrialization, with IgW1 long form (IgW1L) and IgM1 present in free-swimming skin mucus and IgW1L, IgM1, and IgM2 in terrestrialized skin mucus. Combined, these results indicate an increase in investment in the production of unique immune molecules in P. dolloi skin mucus in response to terrestrialization that likely better protects lungfish against external aggressors found in land.

Histology of juvenile skin of Lepidosiren paradoxa Fitzinger, 1837 (Sarcopterygii, Dipnoi)

“…The skin is recognized as a secondary respiratory tissue in several species (Urbina and Meredith 2014), and in lungfish, epidermal mucus is directly related to respiratory physiology (Heimroth et al 2018).…”

A Comparison of the Innate and Adaptive Immune Systems in Cartilaginous Fish, Ray-Finned Fish, and Lobe-Finned Fish

“…While we hypothesize that skin mucus from both cartilaginous fish and lobe-finned fish contains these compounds as well, it has not been as extensively explored as in teleost fish. Supporting this hypothesis are studies showing that a transcript for a lectin, pentraxin, was found in the skin mucus of the common skate ( Raja kenojei ), while AMPs, including histones and S100 proteins, were found in the skin mucus of the African Lungfish ( Protopterus dolloi ) (20, 22).…”

“…The scales of cartilaginous fish are called placoid scales, also known as denticles (16). The skin of lobe-finned fish contains keratinocytes, granulocytes and B cells (20). Lobe-finned fish have cosmoid scales that includes a layer of dense, lamellar bone called isopedine.…”

“…In addition, the AMP Kenojeninin I, has been isolated from the skin of fermented skate ( R. kenojei ) and was found to have inhibitory effects on Bacillus subtilis, E. coli and Saccharomyces cerevisiae (140). A recent study by Heimroth et al (20) identified an increase in proteins with known antimicrobial function including histones and S100 proteins in skin mucus of the lungfish P. dollo during terrestrialization.…”

Conserved keratin gene clusters in ancient fish: An evolutionary seed for terrestrial adaptation

“…However, it is possible that the "adult epidermal keratin genes" were not expressed in lungfish skin in underwater environments. Indeed, Heimroth et al (2018) reported that the epidermis of lungfish showed notable changes and became more compact with flattened keratinocytes in response to experimental terrestrial conditions. Therefore, it would be worthwhile to analyze the RNA-seq data collected from the skin of this terrestrial condition to examine the above possibility.…”