Effects of gelatinous tissue and specimen damage on TMAO analyses from deep-sea fish

FIGURE: Osmolalities and TMAO contents of fish muscle from Linley et al. 2017 in black/grey symbols, with new previously unpublished data on damaged fish in red.DETAILS: Hydrostatic pressure perturbs protein function, and while many proteins in deep-sea animals have evolved partial pressure resistance, many appear to require piezolytes: small organic molecules which counteract pressure’s perturbing effects on proteins via strengthening water structure in cells. The best known piezolyte, <b>trimethylamine N-oxide (TMAO),</b> has been found to <b>increase with depth--linearly with pressure--in muscle tissue of bony and cartilaginous fishes, squids, cnidarians and crustaceans</b>. For hadal fishes including the Mariana snailfish (Linley et al. 2017) and for amphipods (Downing et al. 2018), the linear trend extends all the way down to the greatest ocean depths at which these animals are found (reviewed by Yancey 2020, 2023). However <b>two recent studies have reported considerably lower TMAO levels</b> in a few specimens of hadal snailfish in the Yap and Mariana trenches, plus an abyssal cusk eel (Mu et al. 2021; Xu et al. 2025), suggesting that TMAO does not increase linearly with depths below about 4000 m in bony fishes. While these data may be accurate (though Xu et al. did not include our Mariana grenadier and snailfish data from Linley et al. 2017), they might instead be skewed lower due to 1) gelatinous tissue embedded in muscles, 2) physical damage to fish retrieved into very warm tropical waters over those trenches, and/or 3) TMAO loss due to methodologies.1) <b>Gelatinous tissue</b>: we have shown that such tissue is mostly non-cellular water and has low TMAO, which is found mainly intracellularly in most (though not all) fishes. Therefore gel, when embedded in muscle as we have reported for some cusk eels (<i>Spectrunculus grandis</i>), blob fish (<i>Psychrolutes</i>) and giant grenadiers (<i>Albatrossia pectoralis</i>), can produce much lower TMAO values than found in fishes with more robust muscles, due to cellular TMAO being diluted by watery gel during tissue extraction. However, if this dilution is accounted for by measuring tissue water content and dry mass, TMAO levels inside muscle cells are estimated to be very similar to that in less-gelatinous fish muscle. See Samerotte et al. 2007 and Gerringer et al. 2017 for details.2) <b>Physical damage</b>: during our retrieval of Mariana hadal snailfish, we found that most suffered considerable damage from the capture process including variable times that the lander spent in very warm tropical surface waters, as shown in the FIGURE here. Damage included numerous bloody lesions, and severe lacerations and ruptures in the skin (Gerringer et al. 2017). Note in the FIGURE (red circles) that these damaged fish yielded much lower TMAO levels than we found in intact fish (dark grey circles), and moreover that the damaged fish had higher NaCl levels. Thus, damage seems likely to have resulted in leakage of TMAO from, and infiltration of seawater into, the muscles underlying the gel layer.3) <b>Tissue handling</b>: First, TMAO degrades unless tissues are rapidly frozen after retrieval in liquid nitrogen or dry ice and stored at or below -80˚C on the ship. We do not know how the recently analyzed specimens were handled in this regard. Second, TMAO can potentially be lost during tissue purification; e.g., we found that TMAO binds strongly to some types of ion-exchange resins; such resins are used to purify tissue extracts in some methodologies (Xu et al. 2025), though we do not know if those used actually retain TMAO. In contrast, Xu et al. 2025 suggest that our picric-acid spectrophotometric method for TMAO analysis (as opposed to their LC/MS method) might yield incorrectly high values due to reactions with other substances (see below), though there is no evidence for this, and TMAO-depth increases have been reported by other labs using non-spectrophotometric methods.Finally, these recent low-TMAO data account for neither i) the increased number of <b>TMAO-producing enzyme genes</b> in the Yap Trench snailfish (Mu et al. 2021); nor ii) the linear increase with depth of internal <b>osmolality</b> documented in much of our previous research from 1996 to 2017 (see FIGURE; reviewed by Yancey 2020); TMAO precisely accounts for that osmolality. Nevertheless, we do acknowledge that these low-TMAO data could be accurate, suggesting alterations are needed to the piezolyte hypothesis; e.g., perhaps diet is the primary source of TMAO with some populations of hadal fish having high intake and others having less; or, if TMAO does not actually increase in the same way as osmolality with depth, then it seems that some other potential piezolyte substance must be increasing with depth (it is not NaCl in specimens in good condition).Downing A.B., Wallace G.T. , Yancey P.H. (2018). Organic osmolytes of amphipods from littoral to hadal zones: Increases with depth in trimethylamine N-oxide, scyllo-inositol and other potential pressure counteractants. Deep-Sea Res. I 138: 1-10 https://doi.org/10.1016/j.dsr.2018.05.008Gerringer M.E., Drazen J.C., Summers, A.P., Linley T.D., Jamieson A.J., Yancey P.H. (2017). Distribution, composition, and functions of gelatinous tissues in deep-sea fishes. Royal Soc. Open Sci. 4: 171063 https://doi.org/10.1098/rsos.171063Linley T., Gerringer M, Yancey P.H., Drazen J.C. , Weinstock C., Jamieson A. (2016). Fishes of the hadal zone including new species, in situ observations and depth records of Liparidae. Deep-Sea Res. I, 114: 99-110. https://doi.org/10.1016/j.dsr.2016.05.003Mu Y., Bian C., Liu R., Wang Y., Shao G., Li J., et al. (2021): Whole genome sequencing of a snailfish from the Yap Trench (~7, 000 m) clarifies the molecular mechanisms underlying adaptation to the deep sea. PLoS Genet 17(5):e1009530 https://doi.org/10.1371/journal.pgen.1009530Samerotte A.L., Drazen J.C., Brand G.L., Seibel B.A., Yancey P.H. (2007). Contents of trimethylamine oxide correlate with depth within as well as among species of teleost fish: an analysis of causation. Phys. Zool. Biochem. 80: 197-208 Xu H., Fang C., Xu W., Wang C., Song Y., Zhu C., et al. (2025). Evolution and genetic adaptation of fishes to the deep sea. 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