Effects of fixatives on stable isotopes of fish muscle tissue: implications for trophic studies on preserved specimens

Abstract

Isotopic ecology has been widely used to understand spatial connectivity and trophic interactions in marine systems. However, its potential for monitoring an ecosystem's health and function has been hampered by the lack of consistent sample storage and long-term studies. Preserved specimens from museum collections are a valuable source of tissue for analyses from ancient and pre-modern times, but isotopic signatures are known to be affected by commonly used fixatives. The aim of the present study was to understand the effects of fixatives on isotopic signatures of bulk tissue (d 13 C m and d 15 N m ) and amino acids (d 13 C AA and d 15 N AA ) of fish muscle and to provide correction equations for the isotopic shifts. Two specimens of each: blue cod (Parapercis colias), blue warehou (Seriolella brama), and king salmon (Oncorhynchus tschawytscha) were sampled at five locations along their dorsal musculature, at four time periods: (1) fresh, (2) after 1 month preserved in formalin, and after (3) 3 and (4) 12 months fixed in either ethanol or isopropanol. Lipid content was positively correlated with C: N ratio (r² = 0.83) and had a significant effect on d 13 C after treatments, but not on d 15 N. C:N ratio (for d 13 C m ) and percent N (for d 15 N m ) from preserved specimens contributed to the most parsimonious mixed models, which explained 79% of the variation due to fixation and preservation for d 13 C and 81% for d 15 N. d 13 C AA were generally not affected by fixatives and preservatives, while most d 15 N AA showed different signatures between treatments. d 15 N AA variations did not affect the magnitude of differences between amino acids, allowing scientists to retrieve ecological information (e.g., trophic level) independently of time under preservation. Corrections were applied to the raw data of the experiment, highlighting the importance of d 13 C m and d 15 N m correction when fish muscle tissues from wet collections are compared to fresh samples. Our results make it possible to retrieve d 13 C m , d 15 N m , d 13 C AA , and d 15 N AA from museum specimens and can be applied to some of the fundamental questions in ecology, such as trophic baseline shifts and changes in community's food web structure through time.Key words: amino acids; environmental change; food web; preserved fish; stable isotopes; trophic ecology. that the effect of a stressor on a given response variable will be greater than the inherent spatial heterogeneity of Manuscript

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“…Muscle tissues (1 cm 3 ) from the dorsal musculature were collected from each fish for analysis. Prior to further preparation, muscle tissues sampled from museum collections were left in deionized water for 1 wk to allow excess preservatives to be washed out of the samples (Durante et al 2020b). Fish muscle tissues were then oven-dried (60°C for 72 h) and subsequently ground with a mortar and pestle until yielding a fine and homogenous powder.…”

“…Isotope results from preserved specimens were corrected for the effects of fixation, preservation, and differences in lipid content (for δ 13 C) using the equations from Durante et al (2020b). These equations can be used for specimens fixed in formalin and preserved in ethanol or isopropanol for long periods of time and take into account the lipid content (parametrized by the C:N ratio) and proportion of N in the fish muscle tissue:…”

“…Here, we can anticipate some variation in accuracy using the correction Eqs. (1) and (2) on samples of O. pullus, as (1) chemical preservation effects are dependent on lipid concentrations and compositions, and thus the C:N ratio of the samples (Kelly et al 2006); (2) herbivorous fish have higher C:N ratio than omnivorous or carnivorous fishes (Mill et al 2007); and (3) the equation from Durante et al (2020b) was not developed using herbivorous fishes. Consequently, it is likely that the increase in δ 13 C Bulk values observed for O. pullus were directly linked to the nonadapted corrections made to the values.…”

“…Recent studies have indicated that it is possible to use fixed/ preserved museum specimens to reconstruct past trends in food web structure (Hannides et al 2009, Hetherington et al 2019. Indeed, δ 13 C AA used to track organic matter sources are not affected by fixatives or time under fixation (Chua et al 2020, Durante et al 2020b. Conversely, while δ 15 N AA values tend to vary between treatments, the authors found that for most fish, this variation is largely within the precision of the measurements and related to time between specimen collection and fixation.…”

“…Conversely, while δ 15 N AA values tend to vary between treatments, the authors found that for most fish, this variation is largely within the precision of the measurements and related to time between specimen collection and fixation. Moreover, the homogeneity of change of all individual AA values still allows for precise measurements of TP for historic samples (Durante et al 2020b).…”