Computational protocol: Intra-colonial diversity in the scleractinian coral, Acropora millepora: identifying the nutritional gradients underlying physiological integration and compartmentalised functioning

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Protocol publication

[…] Zooxanthellae were extracted using the air-spraying technique (). Tissue was removed from the coral skeleton using a jet of high-pressure air from a hand gun (80 psi, 1 cm distance to coral). All sprayed tissue was captured in a thick, polyethylene bag containing 10 ml of ultrafiltered seawater (0.04 mm filtration). The tissue slurry was then poured from the plastic bag into a falcon tube, and the bag was double-rinsed with ultrafiltered seawater that was also collected. The slurry was then homogenised for 20 s (Ultra-Turrax T10B; IKA Labortechnik, Staufenim Breisgau, Germany). A 500 uL aliquot of tissue slurry was taken and combined with a 500 uL aliquot of 3% formalin: filtered seawater for preservation. Zooxanthellae were counted in triplicate using a haemocytometer. Zooxanthellae densities were then standardised to branch surface area (cm2), which was obtained through the simple geometry technique, which has been shown to be suitable for Acropora spp. (), using the software Fiji ImageJ (). [...] Data were analysed using R software version 2.3.1 (; ). Due to non-normality and heteroscedasticity (detected via Shapiro–Wilk and Levene’s tests, respectively), as well as some negative values, data were transformed using a Yeo-Johnson power transformation (caret package ()). Transformed data were then analysed using a one-way analysis of variance (ANOVA) for each parameter measured. Where statistical differences were detected, a TukeyHSD post-hoc test was employed at a significance level of P < 0.05 (agricolae package ()). FA profiles (mg g lipid−1) were also analysed using a linear discriminant analysis (LDA), in order to visualize the relationships between colony position (MASS package ()). An LDA biplot was included to show the top fifteen FA driving the differences between colony position. The colour gradient of the vectors show percentage contribution to LDA loadings. Ellipses show 95% confidence intervals. Figures were prepared using the ggplot2 package (). […]

Pipeline specifications

Software tools ImageJ, Ggplot2
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Calcinosis
Chemicals Fatty Acids