Immobilisation of living coral embryos and larvae

Abstract

Embedding and immobilisation of living cells and microorganisms is used in a variety of research and commercial applications. Here we report the successful extended immobilisation of coral larvae in a low-gelling temperature agarose. Embryos and larvae of five broadcast-spawning Scleractinian species were immobilised in agarose gel and tested in a series of exploratory survival and settlement assays. The optimal developmental stage for immobilisation was after ciliation at approximately 24 hours post-fertilisation, after which, survival of immobilised larvae of all species was nearly 100%. In long-term assays, 50% of Montipora digitata larvae survived immobilised for 89 days. Furthermore, immobilised larvae of multiple species, that were released from the agarose, generally remained capable of settlement. These results demonstrate that the immobilisation of the early life-history stages of corals is possible for a variety of applications in basic and applied science.

Figure 1

Montipora digitata 4-cell embryos immediately (a), 24 hours after (b), and 7 days after (c) embedding. Acropora millepora larvae immediately (d), 24 hours after (e), and 4 days after (f) embedding. Acropora millepora spat immediately after (g), 4 days after (h), and 7 days after (i) embedding. (j) Montipora digitata metamorphosed in gel matrix and imaged using fluorescence microscopy. (k) Platygyra daedalea in gel matrix immediately after embedding. (l) Montipora digitata settled on a rubble fragment after 89 days immobilised. Scale bars = 0.5 mm.

Figure 2

Survival of agarose-embedded Montipora digitata embryos (points are jittered horizontally for clarity) plotted against hours after fertilisation with a logistic model fitted to the data (black curve). Blue shading represents the 95% confidence interval around the model. Red curve indicates the indicative buoyancy of embryos/larvae for many coral species. Grey shading indicates optimal theoretical window for embedding from 19–24 hr corresponding with an estimated 95–99% survival and high larval buoyancy. Model coefficients are presented in the inset table. SE = standard error; OR = odds ratio.

Figure 3

(a) Long-term survival of agarose-embedded and control Montipora digitata larvae in individual wells of 48-well plates (n = 48 larvae per treatment). (b) Average percent survival of agarose-embedded and control larvae tested in 3– to 6–day assays. (c) Average percent settlement of previously embedded and control larvae tested in 24–48 hr assays. * indicates statistically significant differences (p ≤ 0.05) between treatments for a given species.