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# Testing of how and why the Terpios hoshinota sponge kills stony corals

### Experiment 1: Sponge fragments

Evidence of bleaching first occurred 3 days after the treatment and was only evident in the group with fragments of T. hoshinota. No bleaching was detected in the other 2 groups with the black cloth (to block light) and white cloth (control) (Table 1). Chi-square tests confirmed that the occurrence of bleaching depended on the treatments (p < 0.001 in both tests: sponge fragment vs. black cloth, and sponge fragment vs. white cloth).

### Experiment 2: Sponge mixture

Significant difference in CICI was detected among the 3 treatments (p < 0.001; Friedman Test, n = 19; Fig. 1). The treatment with sponge and a black cap had the greatest effect, whereas the treatment with the transparent cap demonstrated the smallest response. All 3 pairwise comparisons were significant [CICI (sponge and black caps) > CICI (black caps) > CICI (transparent caps), n = 19, p < 0.01 in all 3 pair-wise comparisons, Wilcoxon signed-rank tests].

### Experiment 3: Sponge supernatant

Two days after the treatment, a significant difference was detected between the sponge + black cap, and fish-meat + black cap groups (Fig. 2) with the former showing significantly greater effects.

Significant difference in CICI was observed among the 3 treatments, i.e., only the sponge supernatant, only black caps, and both sponge supernatant and black caps (n = 17; p < 0.01, Friedman Test, Fig. 3), 4 days after the treatment. Further pair-wise analyses indicated that both factors combined had stronger effects than when single factors were applied [p = 0.055 (against the black cap alone), p < 0.01 (against sponge alone), Wilcoxon signed-rank test, n = 17]. No significant difference was observed between the 2 single factors (p = 0.12; Wilcoxon signed-rank test, n = 17).

### Stable isotope experiment

Six pieces of transplanted corals, 3 from the isotope labeling group, and 3 from the control group, which had been covered by T. hoshinota were successfully retrieved. The other samples either lost their labels or were not covered by the sponge in the field.

The control corals had δ13C: − 14.4 ± 1.2‰, δ15N: 4.8 ± 0.6‰ (n = 3). The error terms were 95% confidence intervals of the means throughout this study. The sponges grown on the control corals had lower values, i.e., δ13C: − 21.4 ± 0.8‰, δ15N: 3.6 ± 0.1‰ (n = 3). The control corals had a significantly higher heavy stable isotopes content than the sponges grown on them (δ13C, p < 0.01; δ15N; p < 0.05; t-tests, n = 3, Table 2).

The enriched corals had δ13C: 31.8 ± 12.1‰, δ15N: 71.0 ± 29.5‰, and the T. hoshinota grown on them had lower values: δ13C: − 16.0 ± 1.0‰, δ15N: 14.7 ± 5.3‰ (n = 3). In fact, the stable isotope composition of Terpios on the enriched corals was closer to the isotope compositions of the sponges on the control corals, than to the coral tissues underneath them (Fig. 4).

Analysis of the sponges indicated that a significant difference in δ13C along the growth axis was evident only in the samples with the enriched treatment (ANOVA, F2,6 = 8.33; p < 0.05), but not in the sponges grown on the control corals (ANOVA, F2,6 = 0.01; p = 0.99; Fig. 5). A Fisher’s PLSD test indicated that the sponge δ13C was the highest directly on top of the enriched corals, intermediate at the junction, and the lowest at the > 5-cm positions (p < 0.01; n = 3), and the > 5-cm and the < 5-cm positions were not significantly different (p = 0.13; n = 3). In comparing the sponge δ13C between the enriched and control corals of the equivalent positions, a significant difference was only observed in the positions right above the transplanted corals (Fig. 5). This was a clear indication that the incorporated heavy stable C of the sponge was not translocated to the more proximal part of the sponges.

Concerning δ15N in the control group, significant difference was observed among the different positions of the sponge (ANOVA, F2,6 = 6.93; p < 0.05), although the difference was small. Lighter nitrogen was discovered near the expanding fronts (Fisher’s PLSD test, p = 0.01; n = 3; Fig. 6). In the enriched group, significant difference was observed among different positions of the sponge (ANOVA, F2,6 = 6.91; p < 0.01), but the trend was the opposite because the expanding fronts that covered the coral had higher levels of nitrogen. The junction was heavier than the more proximal part. By comparing the δ15N that grew on the 2 groups of corals, the sponges on the enriched corals had higher δ15N than those on the control corals in the newly grown tissues. However, the 2 groups of sponge tissues did not vary significantly in the more proximal parts that were far from the new growth (Figs. 5, 6). This result was consistent with the result of δ13C and did not support the suggestion that the frontal sponge tissues translocate acquired new materials to more proximate parts of the sponge.

### Estimation of underlying coral tissue contribution to sponge

Three possible sources contributed to the stable isotope compositions of the T. hoshinota sponge, namely the coral tissues underneath, microbes and food particles filtered from the water column, and the translocation of tissues or materials from the proximal part of the sponge. The stable isotope of the sponge could be expressed as the following equation:

$$updelta _{{{text{sponge}}}} =updelta _{{{text{coral}}}} times f_{{{text{coral}}}} +updelta _{{{text{water}}}} times f_{{{text{water}}}} +updelta _{{text{back sponge}}} times f_{{text{back sponge}}}$$

(1)

δsponge, Isotope composition of new T. hoshinota tissues; δcoral, Isotope composition of coral tissues underneath T. hoshinota; δwater, Isotope composition of sponge food in the water column; δback sponge, Isotope composition of the proximal part of T. hoshinota; fcoral, Fraction contributed by underlying coral to new T. hoshinota tissues; fwater, Fraction contributed by food particles in the water column to new T. hoshinota tissues; fback sponge, Fraction contributed by proximal part of the tissue to new T. hoshinota tissues.

$${text{and}},f_{{{text{coral}}}} + f_{{{text{water}}}} + f_{{text{back sponge}}} = , 1$$

Assuming that δwater × fwater + δback sponge × fback sponge remained unchanged between treatments, the formula could be transformed to

$$updelta _{{{text{sponge}}}} = f_{{{text{coral}}}} timesupdelta _{{{text{coral}}}} + {text{Constant}}$$

(2)

Therefore, the fraction of new sponge tissues contributed by the underlying coral tissues (fcoral) could be estimated when the δsponge values under different δcoral were available. Because the constant in Eq. (2) was not known, at least 2 pairs of data were required to estimate fcoral. Using 3 paired samples of enriched coral tissues and the sponge tissues that covered them, the fcoral estimated from δ13C and δ15N were 9.5% and 16.9%, respectively, using regression. In Eq. (2), the enrichment of the heavy isotope composition along the food chain was not considered.

Source: Ecology - nature.com