Effects of ethanol-preservation on stable carbon and nitrogen isotopic signatures in marine predators

Horii, Sachiko; Takahashi, Kazutaka; Furuya, Ken

doi:10.3800/pbr.10.91

Cited by 4 publications

(4 citation statements)

References 27 publications

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“…Such d 15 N enrichment following treatment ET was reported in other fishes and molluscs (Kelly et al, 2006;Sweeting et al, 2006;Syväranta et al, 2011;Liu et al, 2013). Ethanol causing tissue hydrolysis, leaching, and extracting certain constituents containing nitrogen from the muscle tissue in addition to lipids could explain these shifts (Horii et al, 2015;Sarakinos et al, 2002). Furthermore, we discovered that treatment LE resulted in a significant increase in d 15 N values in muscle tissues.…”

Section: Discussionsupporting

confidence: 78%

Effects of ethanol storage and lipid extraction on stable isotope compositions of twelve pelagic predators

Shen

Morritt²,

Gong³

et al. 2023

Front. Mar. Sci.

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Stable isotope analysis (SIA) has proven to be a powerful tool in reconstructing diets and characterizing trophic relationships for pelagic predators. Ethanol has been a common preservative solution for biopsy samples from remote areas and archived collections. It is still under debate whether the effects of ethanol (ET) would bias the trophic interpretation of the stable isotope values. Further, lipid extraction (LE) is becoming more popular as a general treatment for standardization prior to SIA, particularly for investigating intra and interspecific variation of sympatric species, because lipids have lower δ13C values. In this study, the long-term (up to 448 days) effects of treatment ET and combined treatments ET and LE (ET+LE) on stable carbon and nitrogen isotope values (δ13C and δ15N, respectively) of twelve pelagic predators from the open ocean were evaluated. Results showed that compared with control values, δ15N values displayed a positive change (δ15Nmean offset was 0.71 ± 0.56‰) but δ13C values had variable results (δ13Cmean offset was 0.42 ± 0.64‰) among all species following treatment with ET during the first 28 days and then remained stable throughout the experiment. Compared with treatment LE results, no difference was observed in δ13C, δ15N values, and C/N ratios through time following treatment ET+LE. These results indicated that treatment ET may have species-specific effects on stable isotope values, and the shifts from treatment LE could counter the changes caused by treatment ET. In addition, after 28 days of preservation, the values following treatment ET were similar to those following treatment LE in low C/N species (C/N<3.5), which suggested ethanol may also affect some of lipid contents from muscle tissues. Nevertheless, further research is needed to focus on the mechanisms that control changes in stable isotope composition in tissues stored in ethanol. Given the effects on pelagic predators, muscle tissue samples stored in ethanol from the open ocean or a museum after LE treatment could be used to develop SIA.

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Section: Discussionsupporting

confidence: 78%

Effects of ethanol storage and lipid extraction on stable isotope compositions of twelve pelagic predators

Shen

Morritt²,

Gong³

et al. 2023

Front. Mar. Sci.

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“…Ethanol‐based sample preservation is commonly used for genomic DNA samples (Bucklin 2000) but is not suitable for samples used in stable isotope analysis it alters carbon isotopes (Kaehler and Pakhomov 2001; Kelly et al 2006; Barrow et al 2008). Carbon stable isotope values of samples preserved in ethanol tend to be enriched because of lipid extraction by ethanol (Kelly et al 2006; Horii et al 2015). Ethanol also cannot be used as an in situ preservative for sediment trap samples, owing to its producing precipitates in seawater.…”

Section: Microscopic Analysis Carbon and Nitrogen Stable Isotope Analmentioning

confidence: 99%

Effects of Lugol's iodine on long‐term preservation of marine plankton samples for molecular and stable carbon and nitrogen isotope analyses

Sano

Makabe

Kurosawa

et al. 2020

Limnology & Ocean Methods

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Carbon and nitrogen stable isotope and molecular analyses are common tools for marine ecological and biological research. However, it is difficult to fix samples for both analyses using only one preservative. A solution of potassium iodide and iodine (known as Lugol's iodine solution) is considered to be a suitable preservative; however, the effectiveness of Lugol's iodine solution for long-term preservation of samples for stable isotope analysis and molecular analysis has not yet been investigated. We tested the effectiveness of 5% and 10% Lugol's iodine solutions by comparing them with marine plankton samples preserved in a 5% formalin solution, as well as preserved by freezing. Although δ 13 C and δ 15 N values of samples fixed in 5% formalin and 5% Lugol's iodine solution were significantly different from those of frozen samples, there were no significant differences in both δ 13 C and δ 15 N values between samples fixed in 10% Lugol's iodine solution and frozen samples, when preserved for a duration from 1 week up to 6 months. Polymerase chain reaction (PCR) assay of samples fixed in 10% Lugol's iodine solution after 18 months of preservation succeeded; however, the DNA in samples fixed in 5% formalin and 5% Lugol's iodine solution could not be amplified using PCR. There was no difference in the sequences of partial 18S rRNA genes between samples fixed in 10% Lugol's solution and frozen samples. Thus, we conclude that 10% Lugol's solution is a suitable fixative for both of carbon and nitrogen stable isotope and molecular analyses.

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“…Approximately 1–10 mg of dried net‐plankton samples was homogenized, and 0.4–2 mg of each sample was measured. We used the dorsal muscles of ethanol‐fixed specimens of micronektonic fish for the isotopic analysis using a method that we previously established with samples collected on the same cruise to correct for the effect of the fixative (Horii, Takahashi, & Furuya, ). After additional extraction treatment with 99.5% ethanol, the stable isotope ratios in fish muscle preserved in ethanol (δ 15 N preserved and δ 13 C preserved ) can be corrected to the original δ 15 N values in frozen samples (δ 15 N corrected ) and δ 13 C values in samples with conventional lipid extraction (δ 13 C corrected ) by the following formulas.

δ^{15} N_{corrected} = 1.04 δ^{15} N_{preserved} - 1.79

…”

Section: Methodsmentioning

confidence: 99%

Stable isotopic evidence for the differential contribution of diazotrophs to the epipelagic grazing food chain in the mid‐Pacific Ocean

Horii

Takahashi

Shiozaki

et al. 2018

Global Ecol Biogeogr

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Aim Biological nitrogen fixation supports primary production in oligotrophic water, but its link to higher trophic levels has not been described fully on a biogeographical basis. Here, we determine the regional patterns of the contribution of the combined nitrogen to biological production within the epipelagic layer of the mid‐Pacific Ocean using the isotopic signatures of nitrogen (δ15N) and carbon (δ13C) in the biological components. Location The mid‐Pacific Ocean along 170° W between the southern subtropical front and the Chukchi Sea. Time period Northern and austral summer in 2013 and 2014. Major taxa studied Planktonic and micronektonic biota in the euphotic layer. Methods We measured the geographical variations in δ15N and δ13C of the suspended particulate organic matter (POM), mesozooplankton assemblage and micronektonic fish. We analysed the relationships among these values and the environmental variables of temperature, nitrate concentration and biological nitrogen fixation activity along a 12,000‐km meridional transect. Results The POM δ15N at 0 m was negatively correlated with in situ N2 fixation activity in the subtropical region, whereas that in the equatorial and high‐latitude regions was correlated with the nitrate concentration at 0 m. We found that the ratios of the increase in δ15N to δ13C along the grazing food chain were consistent throughout the equatorial and subtropical regions. Cluster analyses based on the stable isotopic signatures in the biotic components revealed that the food chains in the stations within the subtropical mid‐Pacific Ocean were separated into three groups based on the differential contributions of biological nitrogen fixation. Main conclusions Distinct food chains from primary to tertiary production sustained by different nitrogen sources, nitrate below the euphotic zone, and diazotrophic nitrogen occur within the same biogeographical provinces in the subtropical mid‐Pacific Ocean. The diazotroph‐dominant community contributes substantially to the apex predators in the central areas of the subtropical gyres.

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Effects of ethanol-preservation on stable carbon and nitrogen isotopic signatures in marine predators

Cited by 4 publications

References 27 publications

Effects of ethanol storage and lipid extraction on stable isotope compositions of twelve pelagic predators

Effects of ethanol storage and lipid extraction on stable isotope compositions of twelve pelagic predators

Effects of Lugol's iodine on long‐term preservation of marine plankton samples for molecular and stable carbon and nitrogen isotope analyses

Stable isotopic evidence for the differential contribution of diazotrophs to the epipelagic grazing food chain in the mid‐Pacific Ocean

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