A review of coral bleaching specimen collection, preservation, and laboratory processing methods

Abstract

Under current climate warming predictions, the future of coral reefs is dire. With projected coral reef decline, it is likely that coral specimens for bleaching research will increasingly become a more limited resource in the future. By adopting a holistic approach through increased collaborations, coral bleaching scientists can maximize a specimen's investigative yield, thus reducing the need to remove more coral material from the reef. Yet to expand a specimen's utility for additional analytic methods, information on how corals are collected is essential as many methods are variably sensitive to upstream handling and processing. In an effort to identify common practices for coral collection, sacrifice, preservation, and processing in coral bleaching research, we surveyed the literature from the last 6.5 years and created and analyzed the resulting dataset of 171 publications. Since January 2014, at least 21,890 coral specimens were collected for bleaching surveys or bleaching experiments. These specimens spanned 122 species of scleractinian corals where the most frequently sampled were Acropora millepora, Pocillopora damicornis, and Stylophora pistillata. Almost 90% of studies removed fragments from the reef, 6% collected skeletal cores, and 3% collected mucus specimens. The most common methods for sacrificing specimens were snap freezing with liquid nitrogen, chemical preservation (e.g., with ethanol or nucleic acid stabilizing buffer), or airbrushing live fragments. We also characterized 37 distinct methodological pathways from collection to processing of specimens in preparation for a variety of physiological, -omic, microscopy, and imaging analyses. Interestingly, almost half of all studies used only one of six different pathways. These similarities in collection, preservation, and processing methods illustrate that archived coral specimens could be readily shared among researchers for additional analyses. In addition, our review provides a reference for future researchers who are considering which methodological pathway to select to maximize the utility of coral bleaching specimens that they collect.

Keywords: Bleaching; Coral; Experiments; Handling; Methods; Pretreatment; Samples; Sampling; Specimens; Surveys.

Figure 1. Flowchart illustrating the type of methodological information collected from coral bleaching surveys and bleaching experiments.

This includes (1) sampling design, (2) specimen collection, (3) specimen preservation, (4) specimen processing, and (5) downstream analyses. Images hand-drawn by Rowan McLachlan.

Figure 2. The total number of specimens collected during coral bleaching surveys and bleaching experiments published between January 2014 and August 2020 included in this review.

Full details in Table S1.2.6.

Figure 3. Percentage of 171 publications per type of specimens collected from the reef during coral bleaching surveys and bleaching experiments published between January 2014 and August 2020 included in this review.

Full data in Table S1.3.1.

Figure 4. Percentage of 171 publications using each of the specimen sacrificing methods in coral bleaching surveys and bleaching experiments published between January 2014 and August 2020 included in this review.

Figure 5. Downstream analyses conducted in coral bleaching surveys and bleaching experiments published between January 2014 and August 2020 included in this review.

Percentage of 197 publications which conducted downstream analyses in the categories (A) physiology, (B) -omics, and (C) microscopy and imaging HPLC = High-performance liquid chromatography; DMSP/DMSO = dimethyl sulfoniopropionate/dimethyl sulfoxide; MS = mass spectrometry. Additional details in Table S1.6.1.

Figure 6. Overlap in downstream analyses conducted in coral bleaching surveys and bleaching experiments published between January 2014 and August 2020 and included in this review.

Illustrated in the Venn diagram above are the percentage of studies that conducted at least one downstream analysis within each category. For example, 6.6% of studies conducted at least one physiological and one -omic analysis, but none in the category microscopy and imaging.

Figure 7. Heat map summarizing methodological pathways and downstream analyses categorized in this review.

Cells shaded based on number of studies ranging from 1 (pale) to >9 (dark). AB, airbrush; BL, bleach (NaOCl); CRYO, cryopreserved; DMSO, Dimethyl sulfoxide; DMSP/DMSO, dimethyl sulfonio-propionate /dimethyl sulfoxide; DNA, DNA buffer; DNS, did not state; ETH, ethanol; FIX, fixative; FR, frozen with conventional freezer; G, gametes. GLY, glycerol; GR, ground; HPLC, High-performance liquid chromatography; METH, methanol; RNA, RNA buffer; SNAP, snap frozen with liquid nitrogen. The underlying raw data used to construct this table is in the Supplement 2.

Figure 8. Methodological reporting information for coral bleaching surveys and bleaching experiments published between January 2014 and August 2020 included in this review.

Percentage of studies that reported (light blue bars) or did not report (dark blue bars) methodological information relating to (A) sampling design, (B) specimen collection, (C) specimen preservation, and (D) specimen processing. Grey bars show publications for which data was not applicable (e.g., studies which only collected mucus would not report the size of fragment collected). Likewise, not all studies conducted airbrushing or homogenizing during specimen processing.