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

Abstract

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.

Effectiveness of Lugol’s iodine solution for long-term preservation of zooplankton samples for molecular analysis

“…The effectiveness of 10% Lugol preservation in PCRs targeting approximately 400 bp using copepod samples has been reported (Sano et al 2020). The present study demonstrated its effectiveness in PCRs targeting over 1000 bp using various zooplankton taxa.…”

“…Therefore, 10% Lugol is suitable for long-term preservation of a broad range of zooplankton taxa for various molecular analyses. While there was no significant difference in the total amount of DNA extracted from S. danae between 10% Lugol preservation and freezing; the total amount of Examples of zooplankton samples preserved in 10% Lugolʼs iodine seawater (10% Lugol) for 33 months (a-i), samples preserved for 44 months (j-o), bulk zooplankton net-samples (p, q) and color of preservatives in 9 ml vials immediately after preparation (r, t) and after 18 months preservation (s, u, see Sano et al 2020). a: diphyid, b: abylid, c: copepod, d: ostracod, e: doliolid, f: polychaete, g: pteropod, h: euphausiid, i: chaetognath, j-l: Doliolum nationalis, m-o: Scolecithrix danae.…”

“…j and m: preserved in 5% formalin seawater, k and n: preserved in 10% Lugol, l and o: preserved by freezing, p: preserved in 5% Lugol, k: preserved in 10% Lugol, r and s: 5% Lugol, t and u: 10% Lugol. p and s: samples lost its fixing power after 18 months of preservation (see Sano et al 2020). q and u: samples kept their fixing power after 18 months of preservation.…”

“…Lugolʼs iodine solution is often used for the preservation of phytoplankton, microzooplankton (Gifford & Caron 2000, Kok et al 2012, and mesozooplankton (Knoechel & Steel-Flynn 1989, Koski et al 2005, Jaspers & Carstensen 2009 for microscopic analysis. Sano et al (2020) showed that Lu-golʼs iodine solution can effectively preserve copepod samples for a short target length (ca. 400 bp) PCR-based study after 18 months of preservation.…”

Impact of preservation method and storage period on ribosomal metabarcoding of marine microbes: Implications for remote automated samplings

“…Both reagents have been tested as preservative in automated microbial samplings ( Boeuf et al, 2019 ; Formel et al, 2021 ; Poff et al, 2021 ), but can lead to DNA loss ( Renshaw et al, 2015 ) and are likely unsuitable in remote regions where samples cannot be frozen immediately. Hence, although automated technologies – in particular comparative sampling across different regions – offer exciting perspectives, preservation method and storage time are challenging factors for microbial diversity studies ( Sherr and Sherr, 1993 ; Rissanen et al, 2010 ; Metfies et al, 2017 ; Spens et al, 2017 ; Sano et al, 2020 ; Pratte and Kellogg, 2021 ).…”