**3.2 Materials and methods**

**3.2.1 The sponge study model *Rhabdastrella globostellata* (Carter, 1883)**

Sponge *R. globostellata* is a marine sponge of the family Ancorinidae of Tetractinellida order belonging to the large class of Demospongiae. The sponge is usually about 12-15cm wide, a squat sphere with one or several shallow depressions at the top so that the entire sponge resembles a shallow pot, which gives its name also of yellow pot sponge. The depression is smooth and often has many closely-spaced holes that resemble holes in the cheese. There are bumps on the outside of the 'pot', sometimes forming ridges, its characterized by yellow color in the choanoderm, while the surface is typically dark brown. *R. globostellata* is usually a massive sub globular species that can be cup-shaped in large specimens but a morphotype with a convoluted surface (brain-like) seemed also quite common (Cárdenas et al., 2022). It is found throughout the Indo-Pacific region (World Porifera Database) in shallow-water coral reef environments. It was first described by Henry J. Carter as *Stelleta globostellata* in 1883, named after the *globostellate* shape of its spicules (Latin meaning: "sphere" and stellātus = "star-shaped"). Its skeleton consists of silica spicules characterized by triaene, oxyspheraster, oxaе, and oxyaster (Appendix B – Figure B3.2). In New Caledonia, the distribution of this species is still poorly known (http://lagplon.ird.nc/), and thus the difficulty of finding it outside the study site of Bouraké. Thanks to the first chapter results, this species was found as the most massive and abundant throughout the Bouraké lagoon. Then, this sponge has been chosen as a model to understand their strategies to cope with the extreme environment conditions.

**3.2.2 Experimental design**

During both April 2018 and March 2020, in-situ dark incubations (lasting 1 h) of *R. globostellata* were performed to assess the effects of environmental conditions on its dark respiration, nutrient and organic matter recycling. In March 2020, these measurements were repeated in addition to light incubations to assess the effect on sponge photosynthetic symbionts (i.e., symbiont photosynthesis and sponge respiration, and photosynthetic efficiency of the symbionts in the sponge pinacoderm). In April 2018, seven incubations, using a set of four dark-shaded chambers, were performed during five consecutive days at different tidal phases, close to both low tide (LT; n = 12) and high tide (HT; n = 16). In March 2020, 15 incubations using a set of six chambers were performed over 14 days. This time we performed incubations exactly at high tide (HT; n = 42) and low tide (LT; n = 48). During the first week of fieldwork, seven out of 15 incubations were done in the dark only (HT = 18, and LT = 24), while the remaining eight incubations measured both photosynthesis and dark respiration during both high tide (n = 24) and low tide (n = 24). In these experiments, respiration rates in the dark
in the morning at high tide were first measured, and subsequently using the same individuals, their photosynthesis in the light were measured. In the afternoon, at low tide the photosynthesis first and then dark respiration were measured. This inverse procedure was adopted because during the afternoon the light availability was already lower (337.7 ± 257.7 µmol m⁻² s⁻¹) than during the morning (1176.6 ± 88.4 µmol m⁻² s⁻¹), likely affecting any comparison of the photosynthesis measured during HT in the morning (See Appendix B - Table B3.1 for summary of *in-situ* incubations).

In addition to the *in-situ* incubations, in 2020, sponges fragments (HT; n = 9, LT; n = 9) were also incubated *ex-situ* on board the R/V Alis, to assess their photosynthetic responses by pulse-amplitude modulated fluorometry (PAM) (Genty et al., 1989) under both HT and LT conditions.

### 3.2.3 Incubation set up

For each *in-situ* dark incubation or each pair of incubations (light and dark), a fresh independent (i.e., no sponge fragmentation) *R. globostellata* was used, each of similar volume (n = 96, 232 ± 73 cm³). Sponges were collected attached to substrate by scuba diving at 2–4 m depth at the station B2 (Appendix B -Figure B3.1). Sediment and epibionts on the surface of the sponges were gently removed underwater to ensure that only the sponge respiration was measured (i.e., the holobiont). Each sponge was placed in a transparent Plexiglas benthic chamber (total volume of 6.4 L), which was hermetically closed and connected to a pump and an YSI 600 multiparameter probe (Biscéré et al., 2015). In the chamber system, the pump allows seawater to recirculate at a water flow of 2 L min⁻¹, while the probe recorded the temperature and oxygen concentration every minute, therefore regularly monitoring sponge oxygen consumption (i.e., respiration Rdark) and production (i.e., net photosynthesis Pn, only in 2020). The chambers were dark shaded when measuring the holobiont respiration. During each incubation, which lasted ca. 1 h, one chamber with no sponge was used as a blank (i.e., control) to account for the contribution of photosynthetic organisms and microbial metabolism in the water. All the chambers, connecting tubes, and syringes were cleaned at the end of each day using a solution of 10 % HCl for 4 h and further rinsed in deionized water. This incubation protocol was tested in a preliminary experiment to verify that the duration of each incubation was long enough to detect oxygen depletion and avoid low oxygen concentrations in the chamber.

For the *ex-situ* incubations on 14th March 2020, nine small fragments (3-4 cm in diameter) of *R. globostellata* were collected during low tide, and another set during high tide, from individual, spatially separated donor sponges. Seawater was collected from Bouraké using 2 x 25 L tanks. Samples were transported on board the R/V in individual hermetic plastic bags containing seawater
and immersed in a cooler. Both sponge fragments and water were maintained in a controlled temperature room settled at the *in-situ* temperature until data collection.

### 3.2.4 Data collection

At the beginning (i.e., 5 minutes after the chambers were closed) and at the end of each dark incubation in 2018 and 2020, 450 mL of seawater was collected from each chamber with a syringe to assess changes in nutrient, organic matter, and physical seawater parameters.

During light incubations, photosynthetically active radiation (PAR, 400-700 nm) was measured using a LI-1500 light sensor logger connected to a 30 m long cable and a spherical quantum sensor, which was positioned close to the chambers. The photosynthesis to respiration ratio (P : R<sub>dark</sub>) was calculated as:

$$P: R_{dark} = \frac{P_{g} \times \text{hours of daylight}}{R_{dark} \times 24}$$

with P<sub>g</sub> and R<sub>dark</sub> expressed in µmol O<sub>2</sub> cm<sup>-2</sup> h<sup>-1</sup>. The gross photosynthesis to respiration ratio was calculated based on daily budget of 12 h sunlight (photosynthesis) and 24 h respiration (Wilkinson, 1983).

Consumption (i.e., reduction, negative values) and/or production (i.e., increase, positive values) of nutrients, oxygen and organic matter (i.e., all the parameters measured during the incubations) were calculated as the difference between their concentrations at the beginning and end of the incubations, corrected by the change in concentration occurring in the control chamber, and normalized by the duration of incubation (h), the chamber volume (L), and sponge volume (cm<sup>3</sup>). Sponge volume was calculated using a three-dimensional reconstruction technique. Between 80 and 200 photos of each sponge were taken underwater using a waterproof photo-camera (Nikon AW 130) to cover the sponge whole surface. A reference square (3 cm x 3 cm) was placed close to each sponge to scale the model to its real size. The 3D Zephyr Pro software was then used to calculate the volume of each sponge (see **Appendix B – Section B3.1** for method details). In the *ex-situ* incubations, sponge fragments were dark adapted for 30 minutes (Ralph et al., 1999; Biggerstaff et al., 2015), and the effective quantum yield (F<sub>v</sub>/F<sub>m</sub>) and the relative electron transport rate (rETR) were measured using a Diving-PAM, with the RLC curves function.

### 3.2.5 Seawater analyses

In 2018 and 2020 during the dark incubations, seawater samples were collected at the beginning and end of each *in-situ* incubation to calculate the change in seawater physical and chemical parameters
(nutrient and carbon) during the incubations. In 2018, we measured pH<sub>T</sub> (in total scale), oxygen (DO), temperature, nutrient concentrations (orthosilicic acid [Si(OH)<sub>4</sub>], nitrite + nitrate [NOx], ammonium [NH<sub>4</sub>]<sup>+</sup>, and phosphate [PO<sub>4</sub>]<sup>3-</sup>), bacterial abundance (BA), dissolved organic carbon (DOC), dissolved inorganic carbon (DIC), particulate organic carbon (POC), and particulate organic nitrogen (PON) (see chapitre 2, section 2.2.2.3 for further details of the methods used). In 2020, in addition to the above measurements, we also measured the live particulate organic carbon (LPOC), which is the total amount of bacteria and phytoplankton and is important to better assess the capacity of sponges to recycle organic matter. LPOC was converted into carbon content and used to calculate the consumption/production of carbon discharged by the sponge as detritus (i.e., Detritus = POC - LPOC). In 2020, during the light incubations, we only measured pH<sub>T</sub>, oxygen, and temperature (see Appendix B – Section B3.2 for method details).

3.2.6 Data analysis

Each chamber was considered as a replicate. Because we performed all incubations at the peak of the
tides, and the seawater parameters were fully monitored, we separated all measurements into two tide
categories (HT and LT), and for both 2018 and 2020. In order to test for significant differences in: i)
the environmental conditions at the beginning of the incubations; and ii) the change in the chemical
parameters during the incubations, due to the sponge metabolism, two-way ANOVA, followed by
post hoc Tukey tests (HSD), were performed between high and low tide (HT and LT, respectively),
years (2018 and 2020) and their interactions. ANOVAs were performed after verification of normality
and homogeneity of the data or residuals. In order to test for significant differences between tidal
phases on LPOC, phytoplankton and detritus concentrations collected in 2020, unpaired t-tests were
performed on: i) the environmental conditions at the beginning of the incubations; and ii) the change
in the chemical parameters during the incubations. To better summarize and visualize the different
responses in sponge metabolism between the two years and tidal phases, a principal Component
Analyses (PCA) were performed separately for data collected in 2018 and 2020, and for both HT and
LT using the absolute values of the data after normalization. Furthermore, to investigate the effect of
the seawater composition on metabolic activities, a PCA using Spearman correlation matrix was
performed on data collected in 2020 between the seawater chemical parameters measured at the
beginning of the incubations and the absolute values of their changes (i.e., consumption and
production) during the incubations. In order to assess differences in photosynthesis (Pg), respiration
rates (R<sub>dark</sub>) and their ratios (P:R), as well as differences in photosynthetic efficiency of the
photosymbionts using the maximum electron transport rate (rETR) and the effective quantum yield
(Fv/Fm) during the dark/light incubations, unpaired t-tests were performed between tides (HT and
LT) on data collected in 2020. Data were graphically presented using the generalized additive models (GAM). All analyses were conducted in RV4.1, using stats, factorminer, and vegan packages (R Core Team, 2019).

## 3.3 Results

### 3.3.1 Environmental conditions at the start of the dark incubations

Seawater temperature measured at the beginning of the incubations (chambers with sponges + blanks) was significantly different between years, tides and their interactions (*p* < 0.001; **Appendix B - Table B3.2**), while dissolved oxygen and pH were significantly lower at LT (*p* < 0.001; **Appendix B - Table B3.2**), and not significantly different between years (**Table 3.1**; pH = 0.55, DO *p* = 0.076; **Appendix B - Table B3.2**). For these parameters, the highest average temperature (31.73 ± 0.71°C) and lowest pH and DO (7.56 ± 0.03 pHT units and 3.94 ± 0.23 mg O₂ L⁻¹, respectively) were found at LT in 2020. HSD post hoc comparisons confirmed that seawater temperature did not differ between tides in 2018; but did differ between tides in 2020; and that temperature in 2020 was higher than in 2018 (**Appendix B - Table B3.2**).

<table><caption>**Table 3.1.** Summary of the environmental parameters measured at the beginning of the incubations in 2018 and in 2020, both at HT and LT. Data are expressed as mean ± SD; number of replicates are in brackets; nd means not determined.</caption><thead><tr><th></th><th colspan="2">2018</th><th colspan="2">2020</th></tr><tr><th></th><th>HT (n = 16)</th><th>LT (n = 12)</th><th>HT (n = 18)</th><th>LT (n = 24)</th></tr></thead><tbody><tr><td colspan="5"><strong>Physical parameters</strong></td></tr><tr><td>T (°C)</td><td>26.28 ± 1.35</td><td>26.43 ± 0.74</td><td>29.38 ± 0.13</td><td>31.73 ± 0.71</td></tr><tr><td>pH<sub>T</sub></td><td>7.92 ± 0.18</td><td>7.57 ± 0.09</td><td>8.03 ± 0.01</td><td>7.56 ± 0.03</td></tr><tr><td colspan="5"><strong>Chemical parameters</strong></td></tr><tr><td>DIC (µmol C L<sup>-1</sup>)</td><td>2158.7 ± 83.6</td><td>2376.5 ± 116.1</td><td>2103.5 ± 22.00</td><td>2647.1 ± 33.5</td></tr><tr><td>DO (mg O<sub>2</sub> L<sup>-1</sup>)</td><td>5.54 ± 1.36</td><td>4.26 ± 0.58</td><td>5.66 ± 0.24</td><td>3.94 ± 0.23</td></tr><tr><td>Si(OH)<sub>4</sub> (µmol L<sup>-1</sup>)</td><td>11.81 ± 3.95</td><td>19.37 ± 5.01</td><td>9.15 ± 0.21</td><td>21.03 ± 1.07</td></tr><tr><td>No<sub>x</sub> (µmol L<sup>-1</sup>)</td><td>1.58 ± 1.77</td><td>1.35 ± 0.47</td><td>0.46 ± 0.07</td><td>0.64 ± 0.15</td></tr><tr><td>PO<sub>4</sub><sup>3-</sup> (µmol L<sup>-1</sup>)</td><td>0.35 ± 0.11</td><td>0.35 ± 0.10</td><td>0.33 ± 0.02</td><td>0.35 ± 0.10</td></tr><tr><td>NH<sub>4</sub><sup>+</sup> (µmol L<sup>-1</sup>)</td><td>0.38 ± 0.10</td><td>0.37 ± 0.10</td><td>0.84 ± 0.46</td><td>0.86 ± 0.56</td></tr><tr><td>DOC (µmol C L<sup>-1</sup>)</td><td>93.09 ± 6.55</td><td>129.10 ± 12.77</td><td>130.43 ± 1.69</td><td>219.83 ± 22.68</td></tr><tr><td>POC (µmol C L<sup>-1</sup>)</td><td>29.93 ± 8.44</td><td>35.66 ± 16.13</td><td>16.29 ± 1.42</td><td>37.62 ± 11.80</td></tr><tr><td>PON (µmol N L<sup>-1</sup>)</td><td>1.94 ± 0.46</td><td>2.27 ± 1.14</td><td>1.59 ± 0.14</td><td>2.62 ± 0.69</td></tr><tr><td>BA (x10<sup>8</sup> cells L<sup>-1</sup>)</td><td>2.52 ± 4.44</td><td>3.91 ± 1.04</td><td>12.3 ± 1.11</td><td>13.7 ± 5.18</td></tr><tr><td>LPOC (µmol C L<sup>-1</sup>)</td><td>nd</td><td>nd</td><td>3.92 ± 1.90</td><td>1.01 ± 1.39</td></tr><tr><td>Phyto (x10<sup>6</sup> cells L<sup>-1</sup>)</td><td>nd</td><td>nd</td><td>55.5 ± 6.34</td><td>10.3 ± 4.22</td></tr><tr><td>Detritus (µmol C L<sup>-1</sup>)</td><td>nd</td><td>nd</td><td>14.03 ± 1.64</td><td>37.16 ± 11.75</td></tr></tbody></table>
Chemical parameters, with the exception of PO₄³⁻, significantly differed between years and tides, showing some significant interactions (Table 3.1; Appendix B - Table B3.2). Some parameters differed between years, with bacterial abundance (BA) and NH₄⁺ having lower concentrations in 2018 (*p* < 0.001; **Appendix B - Table B3.2**), and NOₓ higher in 2018 (*p* < 0.001; **Appendix B - Table B3.2**). Most of parameters including DIC, Si(OH)₄, DOC, POC and PON showed significant interactions between tides and years (*p* < 0.05; **Appendix B - Table B3.2**). HSD post hoc comparisons showed that all these parameters had higher concentrations during LT, and for both years, reaching the highest concentrations during LT in 2020 (**Table 3.1**). Both phytoplankton and LPOC showed significant differences between tides (*p* < 0.001; **Appendix B - Table B3.2**), with consistently higher concentrations at HT; while detritus, which mostly depends on the POC concentration, was higher at LT (*p* < 0.001; **Appendix B - Table B3.2**).

### 3.3.2 Metabolic activities of the sponge holobiont in the dark

In the dark, all sponges depleted dissolved oxygen in both 2018 and 2020 (Figure 3.1; **Appendix B - Table B3.3**). ANOVA showed that oxygen consumption significantly differed between years (*p* < 0.001; **Appendix B - Table B3.4**), but not between tides (*p* = 0.293; **Appendix B - Table B3.4**). Mean oxygen consumption was highest during the LT in 2020, and was the lowest during the LT in 2018.

<figure><img src="image_29.png" /><figcaption>**Figure 3.1.** Dissolved oxygen consumption per sponge volume measured during the incubations in the dark in 2018 (in gray) at both low tide (LT; n = 9) and high tide (HT; n = 12), and 2020 (in blue) at both high tide (HT; n = 24) and low tide (LT; n = 28). Boxes are the interquartile range of data (25th and 75th percentiles); the horizontal line is the median, and the whiskers represent the data range (i.e., minimum and maximum).</figcaption></figure>
During the incubations there was a decrease in BA, Si(OH)₄, NH₄⁺, DOC, LPOC and phytoplankton cells, and an increase in PO₄³⁻ and NOₓ (**Figure 3.2; Appendix B - Table B3.3**). Both reductions and increases were found for DIC, POC, and PON (**Appendix B - Table B3.3**). Metabolic parameters were significantly different between years, tide and had significant interactions, without any general common response (**Appendix B - Table B3.4**). DIC and NOₓ only differed significantly between years (*p* < 0.001; **Appendix B - Table B3.4**), while NH₄⁺, Si(OH)₄ and DOC only between tides (*p* < 0.001; **Appendix B - Table B3.4**). BA, POC and PON differed significantly between years and tides (*p* < 0.01; **Appendix B - Table B3.4**), while only Si(OH)₄ and NOₓ showed significant interaction between year and tide (*p* < 0.05; **Appendix B - Table B3.4**). Orthosilicic acid Si(OH)₄ decreased during LT, reaching the highest average consumption in 2018, while NOₓ increased more in 2020 reaching the highest value during HT (**Figure 3.2a, b; Appendix B - Table B3.3**). The highest uptake of DOC was found during LT, while POC was always produced except during LT in 2020 (**Figure 3.2e, f**). Bacteria decreased more during LT in 2020 (**Figure 3.2h**). Detritus, LPOC and phytoplankton were measured only in 2020, where they significantly differed between tides (*p* < 0.05; **Figure 3.3; Appendix B - Table B3.4**). Detritus increased at HT and decreased at LT, while the LPOC and phytoplankton always decreased (i.e., were consumed by the sponge) (*p* < 0.05; **Figure 3.3; Appendix B - Table B3.4**). Phytoplankton was consumed significantly more during HT than LT (*p* < 0.001; **Figure 3.3b; Appendix B - Table B3.4**). Between phytoplankton and bacteria, *Synechococcus* (Syn) was the largest source of carbon at both HT and LT (**Appendix B - Figure B3.4**). Phytoplankton composition showed that at both HT and LT, Syn was the most consumed phytoplankton group (**Appendix B - Table B3.5**).
<figure><img src="image_30.png" /><figcaption>**Figure 3.2.** Sponge nutrients and organic matter consumption/production measured during the incubations during the incubations in the dark at high tide (HT) and low tide (LT) in 2018 and 2020. (a) Si(OH)₄, (b) NOx, (c) PO₄³⁻, (d) NH₄⁺, (e) Dissolved Organic Carbon (DOC), (f) Particulate Organic Carbon (POC), (g) Dissolved Inorganic Carbon (DIC), and (h) Bacteria abundance. Data are the difference between the beginning and the end of the incubations. Boxes represent the interquartile range (25th and 75th percentile); the horizontal line is the median, and the whiskers represent the data range (i.e., minimum and maximum).</figcaption></figure>
<figure><img src="image_31.png" /><figcaption>**Figure 3.3.** Sponge consumption/production measured during the incubations in the dark at HT and LT in 2020. (a) Detritus (POC-LPOC), (b) phytoplankton abundance, (c) Life Particulate Organic Carbon (LPOC). Data are the difference between the beginning and the end of the incubations. Boxes represent the interquartile range (25th and 75th percentile); the horizontal line is the median, and the whiskers represent the data range (i.e., minimum and maximum).</figcaption></figure>

The first two principal components (Dim1 and Dim2) of the PCAs accounted for more than 52% and 55% of the changes in the seawater medium chemistry during the incubations made in 2018 and 2020, respectively (Figure 3.4a, b). Samples clustered into two major groups according to the tide for both years. In both years, a clear difference was found for the two tidal phases and most of the parameters we measured contributed to the LT incubation clusters. In 2018, all parameters except DO, showed positive correlations with Dim1 scores, and were densely plotted on the right-hand side of the plot, where samples were clustered into the LT group. In 2020, only phytoplankton and LPOC showed strong negative correlations with the Dim1 scores and were plotted on the left-hand side of the plot, where samples were clustered into the HT group. All the remaining parameters were positively correlated with Dim1 scores and plotted on the right-hand side of the plot where samples were clustered to the LT group. Among them, detritus, PON, DOC and POC showed the strongest positive correlations and appeared to contribute the most to the variation in sponge metabolism during incubations at LT. Detritus, PON, DOC and POC were all more highly concentrated at the beginning of the LT incubations (Table 3.1).
<figure><img src="image_32.png" /><figcaption>**Figure 3.4.** Principal Component Analyses (PCA) on the parameters measured in the incubation medium (a) in 2018, (b) in 2020 at both HT and LT. Each dot in red indicates one single sponge incubation metabolism made during HT, while triangles in blue indicates incubations during LT. Arrows indicate the variable and their length approximates the variance of the variables. The ellipses represent the 95% interval confidence. Large triangles or dots indicate multiple incubations at the same point.</figcaption></figure>

PCA on a Spearman correlation matrix between seawater parameters measured at both the beginning of the incubations in 2020, and absolute value changes during the incubations (i.e., consumption and production) showed clear relationships for some parameters (Figure 3.5). The first two principal components (Dim1 and Dim2) accounted for more than 90% of the variation. Most of seawater parameters were positively correlated with Dim1, including DOC, BA, NH₄⁺, and Si(OH)₄, which were correlated with the sponge nutrient utilization of DOC, Si(OH)₄, BA and NH₄⁺, suggesting that their initial concentrations in the seawater affected their uptake. Furthermore, significant positive correlations were also found for these parameters (Appendix B - Figure B3.3), showing higher consumption at LT when higher concentrations in seawater were observed. In the same way, LPOC
and phytoplankton showing higher consumption at HT when they were more abundant in the seawater (Figure 3.5; Appendix B - Figure B3.3). The only exception was NH<sub>4</sub><sup>+</sup>, which showed positive relationship between the initial concentrations and its consumption, but it was not related to a specific tide.

<figure><img src="image_33.png" /><figcaption>**Figure 3.5.** Principal Component Analyses (PCA) on Spearman correlation matrix on data collected in 2020 between the seawater chemical parameters measured at the beginning of the incubations, and the absolute values of their changes (i.e., consumption and production) during the incubations. Each dot indicates the sponge metabolism parameters while arrows indicate the variable, and their length approximates the variance of the variables.</figcaption></figure>

### 3.3.3 Sponge holobiont photosynthetic activity

The photosynthetic rates and photosynthetic efficiency were only measured in 2020, the former in-situ and the latter ex-situ, and at both HT and LT conditions. During the in-situ experiment, HT differed from LT conditions in the seawater pH (7.9 vs 7.4 pH<sub>T</sub> units, respectively), DO (6.0 vs 3.4 mg O<sub>2</sub> L<sup>-1</sup>, respectively) and temperature (29.9 vs 31.6°C, respectively). However, light irradiance differed considerably between HT and LT, and were 1176.6 ± 88.4, and 337.7 ± 257.7 µmol m<sup>-2</sup> s<sup>-1</sup>, respectively. During the ex-situ incubations, pH was comparable to the in-situ incubations, and temperature was lower than the in-situ incubations at 28.9-29.5°C (Appendix B - Table B3.6).

Photosynthesis and P:R ratios were significantly higher at HT than LT, but dark respiration did not differ between tides (*p* = 0.3381; **Appendix B - Table B3.7; Figure B3.5**). Gross photosynthesis (P<sub>g</sub>) was always positive (due to oxygen production), and higher at HT than at LT (**Table 3.2; Appendix B**)
**B - Figure B3.5; *p* < 0.001; Table B3.7).** P:R ratios at both tide conditions revealed that the oxygen consumption was always higher than its production. The maximum rETR, as well as the initial Fv/Fm measured during the *ex-situ* incubations in 2020 were significantly different between HT and LT. Both rETR and Fv/Fm versus irradiance (PAR) showed lower values during LT (*p* < 0.05; **Figure 3.6; Table 3.2; Appendix B - Table B3.7**).

<table><caption>**Table 3.2.** Summary of the gross photosynthesis (P<sub>g</sub>), respiration (R<sub>dark</sub>), and their ratio (P:R) measured in-situ, and the initial photosynthetic efficiency (Fv/Fm) and rETR<sub>max</sub> measured ex-situ. For in-situ, *n* = 9 and *n* = 8 at HT and LT, respectively; and for ex-situ, *n* = 9. Data are normalized and expressed as mean ± SD.</caption><thead><tr><th></th><th>HT</th><th>LT</th><th>HT</th><th>LT</th><th>HT</th><th>LT</th></tr></thead><tbody><tr><td></td><td colspan="2">R<sub>dark</sub></td><td colspan="2">P<sub>g</sub></td><td colspan="2">P:R</td></tr><tr><td><strong>In-situ</strong></td><td>-1.91 ± 0.19</td><td>-1.79 ± 0.29</td><td>1.25 ± 0.29</td><td>0.38 ± 0.33</td><td>0.32 ± 0.07</td><td>0.10 ± 0.09</td></tr><tr><td></td><td colspan="2">Fv/Fm</td><td colspan="2">rETR<sub>max</sub></td><td colspan="2"></td></tr><tr><td><strong>Ex-situ</strong></td><td>0.5 ± 0.04</td><td>0.5 ± 0.06</td><td>27.6 ± 7.0</td><td>12.6 ± 6.5</td><td></td><td></td></tr></tbody></table>

<figure><img src="image_34.png" /><figcaption>**Figure 3.6.** Generalized additive models (GAM) on (a) relative ETR (rETR) and (b) the effective quantum yield (Fv/Fm) versus irradiance (PAR). Data were obtained from dark-adapted sponges during the ex-situ incubations in 2020 under high tide (HT; *n* = 9 red) and low tide (LT; *n* = 9 black) conditions. The shaded areas indicate the 95% confidence intervals of the GAM model. Individual measurements were also reported.</figcaption></figure>
**3.4 Discussion**

This chapter investigated how the extreme conditions experienced by the massive sponge *R. globostellata* in Bouraké during low tide affect sponge holobiont organic matter and nutrient recycling, and sponge photosynthetic activity. Findings showed different responses between the two years of measurement. In 2018, both at normal (high tide, HT) and extreme (low tide, LT) seawater pH and dissolved oxygen values, our sponge always consumed DOC, and produced POC (i.e., the sponge loop pathway). In contrast, in 2020 the organic matter recycling was altered, because although sponges always consumed DOC, they produced less POC and detritus at high tide, and stopped (i.e., negative values) producing both at low tide. These differences in 2020 occurred at the same seawater pH, and dissolved oxygen values as measured in 2018, but in 2020 the temperature was much higher, being the highest ever recorded at Bouraké. Similarly, photosymbiont photosynthetic activity was negatively affected during low tide in 2020. Results suggests that while the combination of extreme levels of acidification and deoxygenation seem not to affect *R. globostellata* metabolism and the sponge loop, temperature may be a key driver in metabolic shifts, likely when combined with extreme levels of acidification and deoxygenation.

**3.4.1 Changes in sponge organic matter recycling**

Results of this chapter demonstrate that although the values of acidification and deoxygenation at Bouraké are extreme, the massive sponge *R. globostellata* preferentially produces detritus, which is a prerequisite for the sponge loop pathway. All of the performed incubations under both normal (high tide = HT) and extreme conditions (low tide = LT) confirmed the first step of the sponge loop, i.e., sponge DOC uptake, which is in agreement with the trend reported for many other species, both encrusting and emergent forms (e.g., Yahel et al., 2003; Hoer et al., 2018; McMurray et al., 2018; Wooster et al., 2019), and recently reported for the same study species (Hildebrand et al., 2022). In addition, a positive correlation between DOC consumption and its initial concentration was found (**Appendix B - Figure B3.3**), as also found in previous studies (i.e., Mueller et al., 2014; McMurray et al., 2016; Morganti et al., 2017; McMurray et al., 2018). However, DOC uptake at low tide in 2020 was not higher than in 2018 (**Figure 3.3e**), even though the amount of available organic matter was almost double in 2020 (129.10 vs 219.83 µmol C L⁻¹). This could suggest that the positive relationship between DOC uptake and DOC availability (as in Archer et al., 2017) only exists up to a certain DOC level, which likely corresponds to the optimum organic matter uptake for the sponge.
Furthermore, all the sponges incubated in 2018 and the ones incubated at HT in 2020 produced both POC and/or detritus (Figure 3.2f, Figure 3.3a; Appendix B - Table B3.3), therefore also confirming the second step of the sponge loop, i.e., DOM transformation to both POC and detritus (e.g., De Goeij et al., 2013; Alexander et al., 2014; Rix et al., 2018; Kornder et al., 2022). The reported detritus production (in average 0.06 µmol h⁻¹ cm⁻³) was higher compared to the majority of massive sponges in the Caribbean for which only small amounts of detritus production were reported (< 0.032 µmol h⁻¹ cm⁻³, from McMurray et al., 2018). While DOM transformation is common in encrusting and deep-sea sponges from the Caribbean and Red Sea, with net detritus production found for 19 out 22 species (reviewed by de Goeij et al., 2017), to the best of my knowledge, this study is the first demonstrating high detritus production in a massive sponge in shallow-waters from the Pacific Ocean (Appendix B - Table B3.3).

In contrast, during the LT (extreme conditions) in 2020 (Figure 3.2f, Figure 3.3a, Appendix B - Table B3.3), a negative values of both POC and detritus were found, which means that neither POC nor detritus were produced by the sponges. This result is consistent with recent studies reporting examples of emergent sponges that consumed detritus. For example, the giant barrel sponge *Xestospongia muta* consumed 0.11 ± 0.16 µmol h⁻¹ cm⁻³ in Key Largo, Florida, and 0.016 ± 0.009 µmol h⁻¹ cm⁻³ in Belize of detritus (McMurray et al., 2018). The causes of the disruption of detritus production during low tide in 2020, and the consequent interruption of the sponge loop, could be the result of the extreme physical conditions in pH, DO, and temperature. When considering only the condition and results from 2020, we are unable to disentangle the contribution of the single parameters on the sponge loop since pH, temperature, and dissolved oxygen are significantly correlated with the detritus production/consumption (Appendix B - Figure B3.6), and all were negative at LT. Based on these results, I conclude that the combination of these extreme parameters may have caused the disruption of the second step of the sponge loop in *R. globostellata* during the LT in 2020.

The regression between POC and the main physical parameters measured at the beginning of the incubation at both 2018 and 2020 was also analysed. Indeed, since POC is mainly composed of detritus, as LPOC comprises a maximum of 24 % of POC (Table 3.1), POC production could be a good proxy to assess the second step of the sponge loop, and to compare data between years (Appendix B - Figure B3.7). In this case, temperature seem to be the main factors driving the reduction in POC (i.e., detritus) production, clearly showing that while POC was always produced in 2018 at temperature of ca 26-28 °C, it was mostly consumed during the low tide in 2020 at
temperature of ca 31–33 °C (p < 0.001, **Appendix B - Figure B3.7**). Further laboratory experiments are needed to confirm the effect of temperature on sponge loop pathway.

Another possible explanation for the change in the detritus production measured during the LT in 2020, is a reduction in sponge pumping rates as demonstrated in previous studies (e.g., Massaro et al., 2012; Stevenson et al., 2020). However, if there was a halt in pumping rates during extreme conditions, sponges would simply stop recycling nutrients and carbon, which was not the case. Although I did not measure this parameter, data showed that even at LT in 2020 sponges continued to recycle DOC, as well as other nutrients (**Figure 3.2**), and respired at the same rates as at HT in the same year, therefore the sponge was not stressed and had a normal basal metabolism (**Figure 3.1; Appendix B - Table B2.3**). However, sponge respiration rates were higher in 2020 than in 2018 (**Appendix B - Table B3.3**), possibly as a mechanisms to cope with the high temperatures (**Table 3.1**).

I also hypothesize that this break in the detritus production at LT in 2020 was due to the lower concentration of LPOC and phytoplankton found at LT (**Table 2.1; Figure 3.5; Appendix B - Figure B3.3**), while both were more abundant and more consumed during HT. However, given LPOC was a very small part of the POC pool, the POC mainly comprised detritus (**Figure 3.2f, Figure 3.3a**), and both POC and detritus were always higher at LT (**Table 3.1**), therefore available for the sponge nutrition. Although 1h incubations were too short to measure the production of ‘waste’ filtering cells, which take place every five to six hours (De Goeij et al., 2013), both at HT and LT in 2018 and HT in 2020 *R. globostellata* produced POC (**Figure 3.2f, Figure 3.3a**) but this stopped at LT in 2020.

The finding that sponges stopped producing POC and detritus during the extreme conditions measured at LT in 2020 means that they reduced cell turnover, which is energetically costly and may represent 75 % of the daily energy (DOC and POC) consumed (Kahn and Leys, 2016). Lower detritus production was also found in mesophotic sponges where it is seems that sponges preferentially invest in their growth rather than in cell turnover, when compared with the same species in shallower water (Hadas et al., 2009; De Goeij et al., 2017; Lesser et al., 2020). This hypothesis, which needs to be experimentally validated with others techniques (e.g., NanoSIMS ans stable isotope analyses), is in agreement with findings for Caribbean massive sponges where less carbon is released as detritus (e.g., Pawlik et al., 2015; 2016, McMurray et al., 2018). Sokolova et al. (2012) also suggested that when food is not limiting, high growth rates and biomass accumulation can be supported even under stressful conditions. This might partially explain why our study species is so massive and abundant across Bouraké, covering up to 40% of the reef area (Maggioni et al., 2021), but is rare and forms small colonies outside the lagoon.
In conclusion, several hypotheses could explain the change in sponge organic matter recycling, and further studies are needed to understand the processes that occur when carbon is no longer released as detritus. However, based on the environmental conditions measured in Bouraké, the reported observations suggest that high temperature, in combination with acidification and deoxygenation, may be the main factor driving the break.

### 3.4.2 Effect of extreme conditions on sponge holobiont nutrient recycling and feeding

Beyond their role in carbon recycling on coral reefs, sponges likely have a key influence on nutrient biogeochemistry (e.g., De Goeij et al., 2017; Pawlik and McMurray, 2020). Sponges are important sources of dissolved inorganic nitrogen (DIN), such as ammonium, nitrite and nitrate; and also phosphate through their metabolism (Jiménez and Ribes, 2007), and through the inextricable activity of their associated microbial communities. Sponges typically remove ammonium from seawater, as a nitrogen source for chemo- and phototrophic bacteria and an energy source for ammonium oxidizing bacteria and archea. For instance, both the sponges *Tethya citrina* and *Xestospongia muta* produced $NO_x$, and the latter consumed $NH_4^+$ (Fiore et al., 2013; López-Acosta et al., 2019), reflecting the diverse nitrogen metabolic pathways within the sponge holobiont (Archer et al., 2017). In agreement with previous findings, and notwithstanding the extreme conditions, a net production of $NO_x$, especially in 2020, and a consumption of $NH_4^+$ were measured (Figure 3.2b, d; Appendix B - Table B3.3). Since both aerobic and anaerobic ammonia-oxidizing microbes have been found in sponges (Bayer et al., 2008), these results could be due to both the photosymbionts and anaerobic activity of the associated bacteria. Microbial symbionts may also sequester a significant amount of phosphorus in the form of polyphosphate (Zhang et al., 2015), however a net, and consistent production of $PO_4^{3-}$ during incubations was found (Figure 3.2c; Appendix B - Tables B3.3; B3.4), which was comparable to the sponge *Tethya citrina* that released 0.005 ± 0.004 µmol h⁻¹ mL⁻¹ of $PO_4^{3-}$ (López-Acosta et al., 2019).

Most sponges rely on silicic acid (DSi), such as $Si(OH)_4$ to produce their silica skeletons and to grow. *Rhabdastrella globostellata* always consumed $Si(OH)_4$, with higher rates during extreme conditions (low tide) when the DSi concentration in Bouraké was the highest (Figure 3.2a; Appendix B - Table B3.3). Average DSi consumption was 0.10 ± 0.043 and 0.057 ± 0.043 µmol h⁻¹ cm⁻³ during low tide in 2018 and 2020 respectively, which corresponded to a $Si(OH)_4$ concentration of 19.37 ± 5.01, and 21.03 ± 1.07 µmol h⁻¹ cm⁻³, respectively. A positive correlation between DSi concentration and uptake was found (Appendix B - Figure B3.3), which is in agreement with other studies where consumption of DSi was reported at a similar rate and correlated with DSi availability (Maldonado et al., 2012;
2020; López-Acosta et al., 2019). The high concentration of DSi during low tide in the Bouraké lagoon could be due to the elevated acidification level (minimum pH<sub>T</sub> = 7.2), which prevents dissolution of Si(OH)<sub>4</sub> and increases its concentration in seawater. This chemical condition results in high DSi availability in the Bouraké lagoon, thus facilitating the skeletal growth of *R. globostellata*.

Although the effect of tide and tidally-driven changes in the seawater plankton and bacterial community have yet to be investigated in Bouraké, our measurements in 2020 clearly showed that the abundance of phytoplankton vs bacteria during the tide had a great effect on sponge metabolism (see chapter 2 for further discussion on the Bouraké functioning). Both the live particulate organic carbon (LPOC) and phytoplankton were more concentrated at high tide (**Table 3.1, Appendix B – Table B3.5**), corresponding to the new water from the open lagoon, while bacteria and detritus concentrations were high at low tide, corresponding to the water coming from the mangrove. Differences in the PCA between 2018 and 2020 was likely because in 2020 the incubations were always done at HT and LT and not close to them as in 2018. In agreement with this diurnal variability in the water quality of Bouraké, *R. globostellata* feeds more on LPOC and phytoplankton during the high tide, and inversely, more on detritus and bacteria during the low tide (**Figure 3.2h, Figure 3.3**). This result is in agreement with a recent study showing that *R. globostellata* efficiently filters bacteria cells (Hildebrand et al., 2022), although less than what we found in Bouraké. This difference may be simply due to the different methods used, or more likely because the Bouraké lagoon is eutrophic (**Table 3.1**) when compared to the study site in Guam by Hildebrand et al. (2022). These bacterial cells are likely a food source (Reiswig, 1975; Ribes et al., 1999) for this sponge, although it is well known that sponges are selective filter feeders preferring the relatively rarer but labile resource of phytoplankton compared to the numerical dominant heterotrophic bacteria (Maldonado et al., 2012; McMurray et al., 2016). In contrast, *R. globostellata* in Bouraké seems to change the food source regardless of the tide. For instance, ingestion rates of both the bacteria and phytoplankton groups increased or shifted from one group to another according to their composition based on the tide (**Appendix B - Table B3.5**). Food selection may result from either passive processes, in which the physical properties of the sponge filter leads to differential uptake of picoplankton cell types, or active processes, in which food selection is mediated by sponge behaviour (Jürgens and DeMott, 1995; Maldonado et al., 2010). Therefore, the abundance and metabolic plasticity that make this species highly competitive in Bouraké could be because food is never limited there, and that the passive filtration method is the one preferably used by *R. globostellata*.
### 3.4.3 Effect of extreme conditions on the sponge photosynthetic activity

Sponges form symbiotic relationships with many microbial groups (e.g., Thomas et al., 2016; Moitinho-Silva et al., 2017b), including some photosynthetic symbionts, which provide photosynthetically-derived carbon to their host (Wilkinson, 1983). Some studies have reported that microbial photosynthetic symbionts are not significantly affected when exposed to both OW and OA (Wisshak et al., 2012; Morrow et al., 2015; Luter et al., 2020, Bell et al., 2022). However, earlier studies showed reduced photosynthetic functioning of associated photosymbionts when exposed to high temperature (Cebrian et al., 2011; Webster et al., 2012; Goodwin et al., 2014; Lesser et al., 2016; Beepat et al., 2020). For example, *Cliona orientalis* showed a significant reduction in photochemical efficiency when exposed to a temperature of 27°C (Ramsby et al., 2018b). Consistent with these earlier studies, *in-situ* measurements in this chapter showed a negative effect of the low tide conditions on sponge symbiont photosynthesis, which corresponded to a drop in the symbiont photosynthetic activity on the sponge pinacoderm measured during the *ex-situ* incubation. In fact, significantly lower values were found during low tide for P_g, P:R, F_v/F_m and rETR, while no differences in respiration was reported during *in-situ* measurements between tides (**Table 3.2, Appendix B - B3.7; Figure B3.5**). This drop might be due to the extreme conditions experienced by the sponges during the low tide, especially seawater pH and temperature, since it has been already demonstrated that some sponges can be tolerant to reduced levels of oxygen (Micaroni et al., 2022). A similar response was observed in the giant barrel sponge (*Xestospongia muta*), where a decrease in the productivity of cyanobacteria symbionts was observed when sponges were exposed to high temperature and low pH (Lesser et al., 2016). The drop in photosynthesis could be due to the differences in the light levels between incubations (i.e., at low tide in the afternoon the light was less than at high tide in the morning). However, both the photosynthetic efficiency and the relative ETR measured on dark-adapted sponges in the lab confirmed the drop in the sponge symbiont photosynthesis between tides (**Figure 3.6; Appendix B - Table B3.7**). Although long-term measurements should be done on the whole energy budget, it seems that even if the symbiosis of *R. globostellata* in Bouraké were greatly affected by the combination of extreme conditions, the effect may not be that large since sponges can rely on heterotrophy, which may plays a greater role in its survival. In fact, *R. globostellata* from Bouraké showed heterotrophy characteristics (P:R ratios < 1.5; **Table 3.2**). To support this hypothesis some studies showed that eutrophic conditions could render bioeroding sponges less dependent on their photosymbionts (i.e., Webb et al., 2017).
**3.5 Conclusion**

In this chapter, the semi-enclosed lagoon of Bouraké as a natural laboratory was used to study for the first time the metabolism and organic carbon recycling in the sponge *R. globostellata* exposed to normal and extreme values in physical and chemical parameters. Results showed that *R. globostellata* can tolerate the combination of low seawater pH and dissolved oxygen during the low tidal phase, and it does not show any metabolic dysfunction in its ability to recycle both the organic matter and nutrients. In contrast, higher temperature coupled with extreme acidification and deoxygenation had a dual effect on the sponge: (i) instead of recycling organic matter into detritus and making it available to higher trophic levels, sponges showed no detritus production; (ii) the photosynthetic activity of the symbionts suffered during periods of thermal stress. On-going laboratory experiments using stable isotope analyses are testing the potential role of temperature as the driver of the break in the detritus production, and the processes that are involved when the detritus production stops. The results of this chapter further shows how important trophic pathways may change under extreme environmental conditions and provides insights into how other sponges may respond to ongoing climate change.
**CHAPTER 4:**

CARBON AND NITROGEN RECYCLING SHIFT IN WARM-
ADAPTED SPONGES

<figure><img src="image_35.png" /></figure>

The experimental set-up at the “Aquarium de Lagon” (Nouméa, New Caledonia), where all *ex-situ*
experimental works were undertaken
## Resumé

Dans le chapitre précédent, j'ai pu étudier les réponses métaboliques de l'éponge *R. globostellata* à différentes conditions environnementales. Le principal résultat a montré un arrêt de la production de détritus pendant les conditions extrêmes, ce qui nous permet d'émettre l'hypothèse que la température serait l'un des principaux facteurs de ce changement dans le recyclage de la matière organique. Compte tenu des résultats obtenus *in-situ*, ce quatrième chapitre s'attache à comprendre les effets de fortes températures dans le recyclage du carbone par l'éponge. La température de surface de la mer pourrait augmenter possiblement jusqu'à +4°C d'ici la fin du siècle, ce qui aura des conséquences négatives sur les écosystèmes coralliens. Dans les récifs coralliens, les éponges jouent un rôle clé dans les processus écologiques en absorbant et en transformant la DOM en la rendant disponible aux autres niveaux trophiques. Jusqu'à présent, les études ont rapporté des effets contrastés, négatifs ou positifs, de la température dans les processus physiologiques des éponges avec des réponses spécifiques aux espèces. Cependant, il n'existe pas d'étude ayant examiné l'effet de la température sur le cycle du carbone et de l'azote des éponges. Dans ce chapitre, une expérience contrôlée en aquarium a été mise en place pour étudier les effets d'une température élevée (32°C) sur les cycles du carbone et de l'azote chez l'espèce dominante de notre site d'étude, i.e. *R. globostellata*. Les individus ont été collectés dans un environnement à température fluctuante (6.5°C par jour) à Bouraké (Nouvelle-Calédonie), où la température atteint en été 33.2°C, et un site contrôle où la température en été est « stable » à 28°C (île Tupeti, Nouvelle-Calédonie). Du mucus corallien (matière organique utilisable par les éponges) marqué au ¹³C et ¹⁵N a été utilisé pour suivre son transfert et son incorporation à la fois dans les tissus des éponges et les détritus relégués pendant des expériences d'incubations, durant lesquelles les changements dans la matière organique de l'eau de mer ont également été mesurés. Les résultats principaux de ce chapitre ont montré que l'incorporation de ¹³C et ¹⁵N au sein des tissus de l'éponge et dans les détritus émis change à 32°C, avec une accumulation plus élevée dans les tissus pour les éponges de Bouraké (adaptées à un environnement extrême). Les résultats montrent que les éponges peuvent s'adapter à des conditions de haute température, possiblement similaires à celles que nous pourrions avoir dans le futur, avec un changement dans l'allocation du carbone et de l'azote entre les tissus et les débris de l'éponge. Enfin, ce travail a mis en évidence que le réchauffement des océans pourrait modifier la manière dont les éponges recyclent le carbone (C) et l'azote (N), donc leur qualité et leur disponibilité dans le récif.

Le travail présenté dans ce chapitre a fait l'objet d'une publication actuellement en cours de révision à la revue *Limnology and Oceanography*.
**Abstract**

Ocean warming will affect the functioning of coral reef ecosystems with unknown cascading effects. We previously found that the massive sponge *Rhabdastrella globostellata* inhabiting the semi-enclosed warm, acidic and deoxygenated lagoon of Bouraké changed the carbon (C) recycling when the environmental conditions were extremes. Any perturbation in the ability of sponges to recycle the dissolved organic matter released by the reef, and make it available to higher trophic levels, might have unknown consequences for the reef trophic chain. In this chapter, using ¹³C- and ¹⁵N-labeled coral mucus, I experimentally demonstrated that high temperature affected the rates of C and nitrogen (N) incorporated in the sponge tissue and released as detritus. Biogeochemical processes were measured in *R. globostellata* from Bouraké, where temperatures reach 33.8°C and fluctuates by 6.5°C on a daily scale, and from a control reef (28°C). Sponges were exposed to constant and variable thermal regimes in an orthogonal experimental design. At the elevated temperature, sponges from Bouraké, which are adapted to elevated and fluctuating temperatures, retained more ¹³C and ¹⁵N in the tissue than they released as detritus, while the control had an opposite response. Such a change in C and N allocation at high temperature, with less detritus being redistributed to the surrounding ecosystem, could be a tradeoff used by the Bouraké sponge population to cope with the extreme environmental conditions, likely altering the availability of essential sources of energy with unknown consequences in the reef trophic interactions.

The work presented in this chapter has been the subject of a publication currently under review at the journal *Limnology and Oceanography*.
**4.1 Introduction**

One of the main effects of climate change is the increase in sea surface temperature causing ocean warming (OW). An increase in seawater temperature will have a cascading effect on marine life and ecological processes worldwide. In coral reefs, sponges play a key role by recirculating carbon and nutrients produced by corals, making these sources of energy available to the surrounding trophic chain (e.g., De Goeij et al., 2013; Rix et al. 2017; 2018; Bart et al., 2021). Although many studies have revealed the physiological stress caused by high temperature on sponges (e.g., Bell et al., 2018b), none of these studies have investigated its effect on the sponges ability to recycle carbon and nitrogen.

The negative effects of OW on sponges have been largely demonstrated on the sponge reproduction (e.g., Abdul Wahab et al., 2014), photosynthetic rates (e.g. Bennett et al., 2017), pumping rates (e.g., Massaro et al., 2012), filtration efficiency, choanocyte chamber size and density (Massaro et al., 2012), and change in their associated microbial composition and functioning (Ramsby et al., 2018a). For example, thermal stress causes the reduction in choanocyte chamber size and density, resulting in a reduced ability to generate new choanocyte chambers and normal or even accelerated shedding of choanocytes (De Goeij et al., 2009). Temperatures higher than 31°C increased the expression of heat shock proteins in some species (López-Legentil et al., 2008; Pantile and Webster, 2011; Guzman and Conaco, 2016). Both acute, short- and long-term exposures to high temperature caused mass mortality events (i.e., Cebrian et al., 2011; Di Camillo et al., 2013; Bell et al., 2017), although other studies showed sponges to be more tolerant to OW than other benthic organisms such as corals (Kelmo et al., 2013). Likely, OW effects on sponges' metabolism are species-specific, and depend on whether a particular species is already near or above its thermal optimum. For instance, species living in thermally variable environments cope better with heat stress when compared to species living in more stable environments (e.g., Oliver and Palumbi, 2011; Rivest et al., 2017).

Thanks to their complex association with microbes, sponges can convert the abundant dissolved organic carbon (DOC) in seawater in particulate organic carbon (POC), and release it as detritus (i.e., sponge loop pathway; De Goeij et al., 2013), and/or store it as biomass (e.g., Pawlik et al., 2016; McMurray et al., 2018). In doing that, sponges play a crucial role as ecosystem engineers since they make available carbon (i.e., energy) to other organisms in the reef, and so link different trophic levels (e.g., De Goeij et al., 2013; Hoytema et al., 2023). Therefore, it becomes crucial to better understand to what extent OW will affect the ability of sponge to recycle the organic matter in coral reef ecosystems.
In the previous field-based study (chapter 3) I found that the sponge *Rhabdastrella globostellata* living in the semi-enclosed lagoon of Bouraké has developed mechanisms to cope with the extreme environmental conditions that characterize this special natural laboratory. During the low tide, Bouraké has environmental conditions ($pH_T$ = -0.8 units; dissolved oxygen = -50%; temperature = +2-3°C) that could be claimed to be close to, or even worse than conditions predicted under the worst climate scenario. Although the harsh conditions, a healthy coral reef was found (Camp et al., 2017; Maggioni et al., 2021). In Bouraké, the sponge *R. globostellata* forms massive colonies, with up to 40% of coverage, while outside Bouraké it becomes rare and forms only scattered, low-size colonies. By incubating sponges *in-situ* at both high (i.e., normal conditions) and low tide (i.e., extreme conditions), I previously found that at extremist condition tested (> 31°C), the sponge organic matter recycling changed from detritus production to no more detritus production in *R. globostellata* (Maggioni et al., 2023). Because the observations were made *in-situ*, where sponges experience a combination of factors, including warming, acidification and deoxygenation, I was unable to elucidate the driver of the observed change. In this chapter, I tested that i) this change was primarily due to the elevated temperature ii) carbon (C) and nitrogen (N) were preferentially stored in sponge tissue (e.g., biomass) than recycled as detritus; and iii) the observed response was specific of sponges from Bouraké. These hypotheses were tested here by comparing the ability to recycle the organic matter (DOC, POC, PON, detritus consumption and, or production) on warm-adapted *R. globostellata* from Bouraké (variable thermal history between 28°C and 33°C), and non-adapted ones from a control reef (constant around 28°C). Enriched ¹³C and ¹⁵N incorporated by corals and released as mucus were used to track the C and N incorporations into the sponge tissue and their transfer as detritus.

## 4.2 Materials and methods

### 4.2.1 Samples collection and maintenance

On the 15th and 17th April 2022, fragments of *R. globostellata* of ca. 5-8 cm long were cut from 50 different mother colonies in Bouraké (west coast of New Caledonia), and at a control reef located in Tupeti Island (Thio, east coast of New Caledonia). While Bouraké is an extreme environment characterized by extreme and fluctuating conditions (Maggioni et al., 2021), Tupeti Island reef is a control site because environmental conditions were assumed to be at the ambient level. At both sites, fragments were collected by scuba diving and immediately fixed on a labelled PVC plate using a wire cable. Plates were then attached to a PVC structure fixed at 2-3 m depth on the reef (Figure 4.1). After three weeks of recovery *in-situ*, sponges showed a complete tissue regeneration where the sponge was cut from the mother colony, and did not show signs of tissue necrosis and discoloration. After the recovery period, sponge fragments were collected at both sites and individually transported
in sterile zip plastic bags. Plastic bags were fully filled with seawater and kept in an icebox during transport. At each site, a Hobo pendant temperature logger recorded each 10 min interval the temperature, which was used to set the average temperature in the acclimation tanks (data not showed). In addition, in Bouraké, sixty fragments of 10-20 cm in diameter of the coral *Acropora tenuis* were collected at 2-4 m depth. Sponges and corals were kept in an icebox and transported within 3 h after collection to the “Aquarium des Lagons” in Nouméa (approximatively 90 km south from Bouraké, and 144 km from the control site) where the experiment was run.

<figure><img src="image_36.png" /><figcaption>**Figure 4.1.** Map of New Caledonia (top panel) with the two study sites and pictures of the fragments of Rhabadastrella globostellata freshly transplanted at the control site (Tupeti Island, **A**), and after one month in-situ in the Buraké lagoon (**B**). Within **A** and **B**, a graph reports a typical temperature variation measured during the summer at each study site. Georep New Caledonia database (https://georep.nc/, last access: November 2022) and Qgis software were used to build the figures.</figcaption></figure>

### 4.2.2 Acclimatization and experimental set-up

Two temperature conditions were used during the experimental set-up: 1) typical summer temperature of 28°C; and 2) fluctuating summer temperature ranging daily from 28.5°C to 33.2°C. These two temperature conditions mimic the *in-situ* conditions measured during the summer 2020 when the temperature values were supposed to affect the *R. globostellata*’s metabolism in Bouraké (Maggioni et al., 2023).
Corals were acclimated and maintained in a unique 120 L tank (flow rates at 2 L min⁻¹) receiving *ca.* 300 µmol photons m⁻² s⁻¹ from two led light systems (Mitras LX6100, GHL Germany) on a 12:12 h light:dark cycle. Two submersible pumps (ON 1000, EHEIM, Germany) mixed seawater in the corals tank. During the acclimation period (two weeks), temperature in the corals tank was increased by 1°C each day from the value measured at the sampling sites (*ca.* 26°C) until reach 28°C, and then maintained at 28°C during the whole experiment.

Sponges were carefully cleaned of any epibionts and distributed into four tanks, two by site (25 sponges each tank of 48 L; flow rates at 800 mL min⁻¹) for acclimatization. Sponges were then gradually acclimated (during one week) to the two temperature conditions of 28°C and variable from 28.5°C to 33.2°C, respectively with one independent tank per site. Each tank was independently supplied with unfiltered and non-stagnant seawater pumped at 3 m depth from the Baie des Citrons (Nouméa, New Caledonia) ensuring at least one complete water renewal every hour. Seawater in the tanks were mixed using a submersible pump (micro-jet MC 320, Aquarium system, USA).

After acclimatization and coral labelling (see below), sponges were randomly divided among the 10 experimental tanks (20 L each) that were positioned close to the coral tank to allow the labelled mucus to be filtered by the sponges. The experimental tanks received an ambient light of *ca.* 10 µmol photons m⁻² s⁻¹, as measured using a NKE PAR with LI-193 spherical quantum sensor. Five tanks were setup at constant temperature (28°C) and contained each four sponges from Bouraké and four from Tupeti Island (Appendix C- Figure C4.1). Five more tanks were setup at variable temperature (28-33.2°C). For each temperature treatment, three tanks were positioned below the coral tank and received directly its seawater, which contained labelled corals (see below), but also could receive independent inflows of water. The remaining four tanks (two for each temperature treatment) received “clean” water (i.e., not from the coral tank) and were used as blanks in the labelling calculations.

Temperature was increased using heaters connected to two Apex controller (Neptune System, USA), which were set-up to either maintain a temperature of 28°C (± 0.06°C), or to simulate the natural temperature fluctuation measured in Bouraké. Seawater temperature was continuously measured by the Neptune System, while pH<sub>T</sub> was measured two times a day in each tank using a portable pH-meter (913, Metrohm) calibrated with TRIS buffer (Dickson lab, batch #T28).

### 4.2.3 Corals labelling

Corals labelling procedure combined methods used by Naumann et al. (2010), and Rix et al. (2018). Corals were enriched with nutritive sources of labelled ¹³C inorganic carbon and ¹⁵N nitrogen by the addition of ¹³C-NaHCO₃ and ¹⁵N-NaNO₃ label compounds (Cambridge Isotope Laboratories, 99%
$^{13}$C and 98% $^{15}$N) during 5 days. Each morning at 8:00, the inflow to the coral tank was stopped and 36 mg L$^{-1}$ $NaH^{13}CO_3$ and 1 mg L$^{-1}$ $Na^{15}NO_3$ were added. The effectiveness of this method was previously tested in a trial experiment (**Appendix C - Table C4.1**). Aquaria pumps maintained water circulation and air exchange for the 8h-labelling period after which the seawater flow-through was resumed. During this period the inflows between the corals tank and the experimental tanks, which were positioned below the coral tank, were closed. Mucus from 15 randomly coral colonies were collected at day 0, 1, and 5 to verify the $^{13}$C and $^{15}$N incorporation. Corals were removed, rinsed in label-free seawater and air exposed for mucus production (2 min). The collected mucus was frozen at -20°C, for $\delta^{13}$C and $\delta^{15}$N determinations.

### 4.2.4 Transfer and incorporation of coral mucus to sponges

At the end of the five days of corals labelling, seawater from the coral tank containing isotopically-labelled *Acropora tenuis* was allowed to continuously feed the six 20 L experimental tanks containing *R. globostellata*, therefore allowing sponges to filter the labelled coral mucus. The flow through from the coral tank to the six tanks were regulated to allow 20 L h$^{-1}$ of water to flow from labelled corals to sponges.

The incorporation of coral mucus ($^{13}$C and $^{15}$N) in sponges' tissue and then to detritus were assessed on labelled and non-labelled sponges, while organic matter recycling (i.e., DOC, POC, PON and detritus fluxes), were assessed on labelled and non-labelled sponges maintained at the two temperature treatments (constant and variable). Since it was technically difficult to maintain the variation in the temperature during the incubation, sponges from the variable treatment were maintained during incubation at the highest temperature at which sponges are exposed. Therefore, sponges were incubated at two different temperatures according to their temperature treatment: i) 28°C as control summer temperature, and ii) 32°C as the highest temperature to which Bouraké sponges were exposed for the longest time. The latter was also chosen since I previously hypothesized that 31°C is the threshold value triggering the break in the carbon recycling for this species (Maggioni et al., 2023). Of the 100 samples collected, only 80 healthy individuals that showed actively seawater pumping were used for the experiment. Indeed, 8 fragments collected at the control site showed signs of bleaching and tissue necrosis when we increased the experimental temperature in the tanks. In contrast, sponges from Bouraké did not show evident signs of stress.

The incorporation of mucus into sponges' tissues (6 labelled tanks + 4 non-labelled tanks) was measured on two specimens each site (n = 2), each tank (n = 10), and from each treatment (n = 2). A total of 40 sponges (24 from labelled tanks + 16 from non-labelled tanks) were incubated after four
days, and the remaining were incubated after 8 days during which sponges received labelled seawater from the coral tank. The collected sponges were removed from the experimental tanks and rinsed in labelled-free flowing seawater during 10 min. Each sponge was then transferred into individual 2 L glass beakers filled with fresh labelled-free seawater and incubated for 3 h. Beakers were semi-submerged in a water bath (n = 4) containing each one 15-places submersible Variomag stirring plate. Each water bath contained a total of 13 incubation beakers (12 with sponges and one with only water that was used as a blank incubation) for labelled tanks, while 9 incubation beakers (8 with sponges and one as a blank incubation) for non-labelled tanks. Sponges from both the control (Tupeti) and variable temperature conditions (Bouraké) were kept respectively at 28°C and at 32°C during the incubation. The incubation was performed in the quasi-dark to avoid the contribution of photosymbionts into the C and N recycling. Temperature in the water bath was verified by the temperature probes connected to the Apex systems. The same procedure was applied to both labelled and non-labelled sponges.

### 4.2.5 Organic matter and seawater chemical measurements

Changes in dissolved organic carbon (DOC), particulate organic carbon (POC), particulate organic nitrogen (PON), detritus, bacteria and phytoplankton were also measured in each beaker as the difference in their contents in the incubation medium between the beginning and the end of each incubation. These changes in organic matter during incubations are considered as a flux resulting from the sponge metabolism. The following measurements were performed on two replicates for each temperature condition at the beginning (water from the inflow alimenting the tanks) and at the end of the incubation (seawater medium on each incubation beaker). Sponges were stored frozen in sterile zip bags at -20°C for $\delta^{13}$C and $\delta^{15}$N analyses, and total C and N content determination. All glass bottles, beakers and vials used were pre-combusted, washed with HCl solutions (10%) and rinsed using milliQ water.

For particulate organic matter (PON and POC), 1.8 L of seawater was filtered into pre-combusted (450°C during 4h) GF/F filters. Filters were then dried at 60°C for 24 h before $^{13}$C and $^{15}$N analyses and total C and N content determination.

For DOC, two pre-combusted (4h at 450°C) glass ampoules were filled with water filtered using a glass syringe filtration system (SGETM) throughout two pre-combusted 25-mm GF/F filters. Samples were then acidified with 70% sulfuric acid ($H_2SO_4$), sealed, and stored at room temperature for dissolved organic carbon analysis by high-temperature catalytic oxidation (HTCO) (Sugimura and Suzuki, 1988; Cauwet, 1994) on a Shimadzu TOC-L analyzer. Typical analytical precision was $\pm$ 0.1-
0.5 µM C (SD). Consensus reference materials (https://hansell-lab.earth.miami.edu/consensus-reference-material/index.html) were injected every 10 to 20 samples to ensure stable operating conditions.

Total bacteria abundance (BA) and phytoplankton abundance were measured on 1.49 mL of water fixed with 0.165 mL of paraformaldehyde (PFA) in cryovials, first kept 10 min in the dark, and then stored at -80°C. The different plankton and BA components were detected and counted using BD AccuriTM C6 cytometer. In this case, phytoplankton were analyzed for 10 min at high flow rate, while bacterioplankton were analyzed for 5 min at high flow rate (see **Appendix B – Section B3.2** for further details of the methods used). In addition, bacteria and phytoplankton (LPOC) in µmol C h⁻¹ g⁻¹ ingested by sponges was used to evaluate their contribution in the C food sources.

Consumption (i.e., reduction; negative values) and/or production (i.e., increase; positive values) of each parameter measured during the incubations were calculated as the difference between their concentrations at the end and at the beginning of incubations. Then, data were corrected by the change in concentrations occurring in the blank chambers (only water), and normalized by the duration of incubation (h), the tank volume (L), and the sponge dry weight (g). Carbon sources were calculated as the percentage of the carbon (DOC, POC, bacteria and phytoplankton) ingested by the sponges. Note that in this case only consumed parameters (negative values) were considered.

### 4.2.6 Isotopic analyses

Coral mucus and sponge subsamples were lyophilized, shredded and homogenized by a mortar and pestle for both labeled and unlabeled samples. Subsamples of sponge tissues, coral mucus and decalcified POM filters were then weighed and transferred into tin capsules for bulk δ¹³C and δ¹⁵N isotope analyses, and total C and N content.

Stable isotope and elemental analyses were conducted using elemental analyzer mass spectrometer Integra CN Sercon at the Mediterranean Institute of Oceanography (Marseille) (Raimbault et al., 2008; Lacoste et al., 2016). The carbon and nitrogen isotopic ratios are expressed in δ¹³C and δ¹⁵N notation (Vienna-PeeDee Belemnite limestone for carbon and atmospheric N₂ for nitrogen) as the deviation from standards in parts per mill, using the following equation:

$$ \delta^{13}\text{C} \text{ and } \delta^{15}\text{N} = [(R \text{ sample}/R \text{ standard}) - 1] \times 1000 $$

where R sample and R standard are the ratio of ¹³C/¹²C or ¹⁵N/¹⁴N in the sample and in the standard reference materials, respectively. R standard C = 0.011118 and R standard N = 0.00368 from
Atmospheric N2. The precisions of replicate analyses were 0.5‰ and 0.3‰ for δ¹⁵N and δ¹³C, respectively.

The incorporation of excess (i.e., background) ¹³C and ¹⁵N is expressed as the specific enrichment, i.e. Δδ¹³C and Δδ¹⁵N values. This was calculated by subtracting the background δ¹³C and δ¹⁵N values of the control sponges from the values of the sponges exposed to labeled corals. The fractional abundance (F) of the heavy isotope in the sample (¹³C/[¹³C + ¹²C] or ¹⁵N/[¹⁵N + ¹⁴N]) was calculated as F sample = R sample/(R sample + 1), and reported as atom percent (atm % = F × 100). The excess fractional abundance of heavy isotope (E) was calculated as the difference between the F sample and F background: E = F sample – F background.

Incorporation rates are the total uptake (I), calculated by multiplying E by the C or N content of the sponge tissue or detritus. To determine the total mucus-derived C (¹²C + ¹³C) and N (¹⁴N + ¹⁵N) incorporated, the total uptake (I) was divided by the fractional abundance of the coral mucus supplied to the sponges. To determine the C:N ratios of coral mucus-derived organic matter incorporated into the sponge tissue and detritus, the total amount of coral mucus-derived C incorporated was divided by the total amount of coral mucus-derived N incorporated. The analytical error was less than 0.13 h for carbon and 0.61 h for nitrogen.

The enrichment in the sponge tissue was graphically represented separately for day 4 and day 8, and pooled together for the statistical analysis. The incorporation of ¹³C and ¹⁵N into the sponge tissues was normalized by the weight of sponges (g⁻¹) and the number of days (day⁻¹). Similarly, the detritus produced during the 3 h incubations was calculated on a day scale assuming regular daily production in the dark. Incorporation values of both sponges' tissues and detritus are expressed in µmol C or N g⁻¹ d⁻¹. The specific fluxes of ¹³C and ¹⁵N were normalized for the number of days (µmol C or N d⁻¹), and expressed as their percentages assimilated in sponge tissue, and excreted as detritus by the sponges.

### 4.2.7 Statistical analyses

General linear model GLM analysis with “tank” as fixed factor was performed to test for a potential tank effect (three tanks per treatment). After verification of the absence of any tank effect, data between the replicated tanks were pooled, and the factor tank was excluded from subsequent models. Since we were not interested to test for the effect of time in the incorporation of ¹³C and ¹⁵N, but we only tested the rates of their incorporation between tissue and detritus, we pooled the data collected during the two incubations, after 4 and 8 days. Significant differences in the organic matter recycle, enrichment, total flow and incorporation, were tested with two-way ANOVAs between temperature
conditions (two levels, 32°C and 28°C), sponges origin (two levels, control and Bouraké), and their
interaction. When the interaction was significant, a Tukey *post hoc* test was performed. ANOVAs
were run after verification of normality and homoscedasticity of the data. All analyses were conducted
in R V4, using stats, factorminer, and vegan packages (R Core Team, 2019).

**4.3 Results**

**4.3.1 Environmental parameters**

Seawater temperature variation settled, and controlled by the Neptune System, matched with the
temperature variability measured *in-situ* during the summer 2020 (Appendix C - Figure C4.1).
Temperatures varied from 28.5°C to 33.2°C in all the tanks for the variable treatment, and from
27.7°C to 28.3°C in all the tanks for the constant treatment, without graphical differences. Seawater
pH<sub>T</sub> were 8.1 ± 0.05 and 8.0 ± 0.07 at the variable and constant treatment, respectively.

**4.3.2 Carbon and nitrogen consumption and production**

Dissolved organic carbon (DOC) was significantly more consumed at 28°C than at 32°C for both
control and Bouraké sponges (**Figure 4.2; Table 4.1a**). Although box plots showed some variability
in the data (all negative skewness), in general all sponges consumed DOC, with the exception of the
control sponges at 32°C, which produced 0.08 ± 2.14 µmol C g⁻¹ h⁻¹ (mean ± SD). Control sponges
at 28°C consumed the highest amount of DOC (**Appendix C - Table C4.2a**). During incubations,
sponges consumed both phytoplankton and bacteria, while produced POC and PON (**Appendix C -
Table C4.2a**). Significant differences were found between temperatures for the bacteria and DOC
consumption, and for the interaction term between temperature and origin for POC, PON (**Table
4.1a**). A significant interaction between temperatures and origins was also found for detritus with the
highest detritus production (i.e., production) and a negative detritus production (i.e., consumption)
measured respectively at 28°C and 32°C for Bouraké (**Table 4.1a, Appendix C - Table C4.2a**).
<table><caption>**Table 4.1.** Summary of two-way ANOVAs between temperature conditions (two levels, constant and variable), origin (two levels, control and Bouraké), and their interaction. **a)** the carbon and nitrogen consumption and/or production measured during 3h of incubation; **b)** carbon sources consumed during the incubations (only data showing carbon consumption were included); **c)** ¹³C and ¹⁵N enrichments (Δδ¹³C and Δδ¹⁵N) measured on both the sponge tissue and detritus at the end of incubations; **d)** ¹³C and ¹⁵N incorporation measured on both tissue and detritus; and **e)** ¹³C:¹⁵N ratio. When the interaction was significant, a Tukey HSD test was applied and only significant differences were reported. **B.** is Bouraké and **C.** the control at 32°C and 28°C.</caption><thead><tr><th></th><th>Temp<br>F-value (P)</th><th>Origin<br>F-value (P)</th><th>Temp x Origin<br>F-value (P)</th><th>HSD Post hoc (P)</th></tr></thead><tbody><tr><td colspan="5"><strong>a) C and N consumption and/or production</strong></td></tr><tr><td>DOC</td><td>6.43 (&lt; 0.05)</td><td>0.006 (0.938)</td><td>0.164 (0.687)</td><td></td></tr><tr><td>POC</td><td>1.865 (0.176)</td><td>0.030 (0.8.3)</td><td>4.369 (&lt; 0.05)</td><td></td></tr><tr><td>PON</td><td>0.436 (0.511)</td><td>0.001 (0.977)</td><td>4.635 (&lt; 0.05)</td><td></td></tr><tr><td>Detritus</td><td>2.518 (0.117)</td><td>0.003 (0.954)</td><td>5.225 (&lt; 0.05)</td><td>B.32°C &lt; B.28°C (&lt; 0.05)</td></tr><tr><td>Bacteria</td><td>17.60 (&lt; 0.001)</td><td>1.27 (0.263)</td><td>0.364 (0.548)</td><td></td></tr><tr><td>Phytoplankton</td><td>0.454 (0.502)</td><td>3.72 (0.06)</td><td>0.428 (0.515)</td><td></td></tr><tr><td colspan="5"><strong>b) Carbon sources</strong></td></tr><tr><td>Bacteria</td><td>19.46 (&lt; 0.001)</td><td>2.63 (0.109)</td><td>2.45 (0.122)</td><td></td></tr><tr><td>DOC</td><td>8.305 (&lt; 0.01)</td><td>0.016 (0.900)</td><td>0.614 (0.438)</td><td></td></tr><tr><td>PON</td><td>8.338 (&lt; 0.01)</td><td>4.464 (&lt; 0.05)</td><td>1.954 (1.170)</td><td></td></tr><tr><td>Phytoplankton</td><td>0.050 (0.824)</td><td>2.107 (0.151)</td><td>1.266 (0.264)</td><td></td></tr><tr><td colspan="5"><strong>c) ¹³C and ¹⁵N enrichment</strong></td></tr><tr><td>¹³C<sub>tissue</sub></td><td>3.841 (0.06)</td><td>0.162 (0.689)</td><td>5.429 (&lt; 0.05)</td><td>B.32°C &gt; C.28°C (&lt; 0.001)<br>C.32°C &lt; B.32°C &gt; C.28°C (&lt; 0.001)</td></tr><tr><td>¹⁵N<sub>tissue</sub></td><td>8.136 (&lt; 0.01)</td><td>20.42 (&lt; 0.001)</td><td>12.14 (&lt; 0.001)</td><td>B.32°C &gt; B28°C (&lt; 0.05)<br>C.32°C &lt; B.32°C (&lt; 0.05)</td></tr><tr><td>¹³C<sub>detritus</sub></td><td>1.403 (0.243)</td><td>0.234 (0.631)</td><td>18.994 (&lt; 0.001)</td><td>B.32°C &gt; B28°C (&lt; 0.05)<br>C.32°C &lt; B.32°C (&lt; 0.05)</td></tr><tr><td>¹⁵N<sub>detritus</sub></td><td>12.85 (&lt; 0.001)</td><td>0.056 (0.814)</td><td>1.558 (0.219)</td><td></td></tr><tr><td colspan="5"><strong>d) ¹³C and ¹⁵N incorporation</strong></td></tr><tr><td>¹³C<sub>tissue</sub></td><td>3.22 (0.07)</td><td>0.767 (0.386)</td><td>14.056 (&lt; 0.001)</td><td>B.32°C &gt; B.28°C (&lt; 0.01)<br>C.28°C &gt; B.28°C (&lt; 0.05)</td></tr><tr><td>¹⁵N<sub>tissue</sub></td><td>8.184 (&lt; 0.001)</td><td>21.454 (&lt; 0.001)</td><td>24.546 (&lt; 0.001)</td><td>B.32°C &gt; B.28°C (&lt; 0.001)<br>C.28°C &gt; B.32°C (&lt; 0.001)<br>C.32°C &lt; B.32°C (&lt; 0.001)</td></tr><tr><td>¹³C<sub>detritus</sub></td><td>10.13 (&lt; 0.01)</td><td>0.933 (0.34)</td><td>3.194 (0.08)</td><td></td></tr><tr><td>¹⁵N<sub>detritus</sub></td><td>13.51 (&lt; 0.001)</td><td>1.968 (0.168)</td><td>2.061 (0.159)</td><td></td></tr><tr><td colspan="5"><strong>e) C:N ratio in sponge tissue and detritus</strong></td></tr><tr><td>C:N<sub>tissue</sub></td><td>1.49 (0.23)</td><td>5.71 (0.05)</td><td>0.265 (0.61)</td><td></td></tr><tr><td>C:N<sub>detritus</sub></td><td>4.24 (0.05)</td><td>2.36 (0.232)</td><td>4.44 (0.05)</td><td>C.32°C &gt; C.28°C (&lt; 0.05)</td></tr></tbody></table>
<figure><img src="image_37.png" /><figcaption>**Figure 4.2.** Sponge dissolved organic carbon (DOC) and detritus consumption/production during the incubations at 28°C and 32°C for both sponges from Bouraké and Control site (Tupeti). Boxes are the interquartile range of data (25th and 75th percentiles); the horizontal line is the median, and the whiskers represent the data range (i.e., minimum and maximum).</figcaption></figure>

To investigate which carbon source were used by the sponges, only the carbon uptake (negative values) was accounted. Despite DOC was the highest carbon source available (Appendix C - Table C4.2c, d), bacteria was the main source of carbon consumed at both temperatures (Figure 4.3). Temperature significantly affected carbon source uptake for bacteria, DOC and POC, the latter also showing significant differences between origins. Consumption was the highest at 32°C for bacteria, and at 28°C for DOC and POC (Table 4.1b; Figure 4.3), while Bouraké sponges consumed more POC at 32°C and control sponges consumed more POC at 28°C (Figure 4.3).
<figure><img src="image_38.png" /><figcaption>**Figure 4.3.** Carbon source uptake (%) by the sponge at 28°C and 32°C from Bouraké and Control (Tupeti) sites. Only consumed values (negative values) of bacteria, dissolved organic carbon (DOC), phytoplankton and particulate organic carbon (POC) as sources of carbon were considered.</figcaption></figure>

### 4.3.3 Sponges and detritus enrichment

After 5 days of incubation with labelled C and N nutritive sources, coral mucus resulted to be enriched on average by 416.5 ± 62.5‰ for δ¹³C and 900.6 ± 185.8‰ for δ¹⁵N (**Appendix C - Tables C4.3**). Isotopic signatures incrementally increased in the sponge tissue between day four and day 8, suggesting gradual accumulations of labelled C and N over time, and finally their transfers as detritus (**Figure 4.4**). Data were pooled between days. Statistical differences were found for the interaction term between temperature and origin for the ¹³C accumulated into the sponge tissue and transferred into detritus (**Table 4.1c**). The detritus ¹⁵N enrichment differed between temperatures, while it differed between temperatures, origins, and their interaction in the sponge’s tissue. In general, both ¹³C and ¹⁵N were highly enriched at 32°C, both in the sponge tissue and into detritus.
<figure><img src="image_39.png" /><figcaption>**Figure 4.4.** Stable isotope enrichment of ¹³C and ¹⁵N in sponge tissue (a-b) and detritus (c-d) after 4 and 8 days of experiment, at 28°C and 32°C, for both sponges from Bouraké (dots) and Control (triangles). Non-enriched stable isotope signature as also reported as CTRL (square). Data as expressed as isotope tracer enrichment Δδ¹³C (‰) and Δδ¹⁵N (‰). Note that detritus enrichment was calculated on a day basis.</figcaption></figure>

#### 4.3.4 ¹³C and ¹⁵N total fluxes and incorporation in the sponge tissue and detritus

Enriched ¹³C and ¹⁵N total fluxes in sponge tissue were significantly different in the interaction between temperature and origin, while detritus only differed between temperatures (Appendix C - Table C4.4). Sponges from Bouraké showed higher ¹³C and ¹⁵N total flux at 32°C for both tissue and detritus compared to 28°C, while control sponges showed higher ¹³C and ¹⁵N total flux at 32°C only
for detritus compared to 28°C (Appendix C - Figure C4.2). Overall, both ¹³C and ¹⁵N labelled fluxes of coral mucus were always more accumulated in sponge tissue (ca 57%, and 65%, respectively) than excreted as detritus (ca 42%, and 34%, respectively), with only an exception for control sponges at 32°C, which showed a balance between the total flow excreted (47% ¹³C, and 52% ¹⁵N) vs accumulated (52% ¹³C, and 47% ¹⁵N) (Appendix C - Figure C4.3). When carbon and nitrogen fluxes were normalized to the sponge weight, both the statistical significances and the sponge responses stay consistent (Table 4.1d; Figure 4.5). Specifically, although significant differences in the interaction of temperatures and origins were found in sponge tissue, detritus showed significant differences only between temperatures with higher incorporation at 32°C for both ¹³C and ¹⁵N (Table 4.1d; Figure 4.5). Incorporation of ¹³C in sponge tissue was higher for the Bouraké sponges at 32°C than at 28°C (30.4 ± 13.9 µmol C g⁻¹ d⁻¹, and 15.1 ± 8.5 µmol C g⁻¹ d⁻¹, respectively), and ¹³C in sponge tissue for the Bouraké sponges was lower than control sponges at 28°C (Table 4.1d; Figure 4.5). The ¹⁵N also showed higher incorporation (16.9 ± 4.7 µmol C g⁻¹ d⁻¹) for Bouraké sponges at 32°C than at 28°C for both control and Bouraké sponges (8.0 ± 2.3 µmol C g⁻¹ d⁻¹, and 7.9 ± 3.1 µmol C g⁻¹ d⁻¹, respectively). In addition, incorporation of ¹⁵N for Bouraké sponges at 32°C was higher than for control sponges at 32°C (Table 4.1d; Figure 4.5).

The C:N ratio in sponge tissues did not significantly differ between temperatures but showed
differences between origins, while its ratio in control detritus at 32°C was significantly higher than
at 28°C (Table 4.1e). The C:N ratio was always double in the sponges tissue than in the detritus
(Appendix C - Figure C4.4), with a total average of 4.53 ± 0.21 and 2.74 ± 1.35, for sponge tissue
and detritus, respectively (all data pooled).
<figure><img src="image_40.png" /><figcaption>**Figure 4.5.** Sponge tissue and detritus incorporation of ¹³C (a) and ¹⁵N (b) at 28°C and 32°C, for both sponges from Bouraké and control site (Tupeti). Daily incorporation rates (mean ± SD) of coral mucus are expressed in µmol C or N g⁻¹ d⁻¹.</figcaption></figure>

**4.4 Discussion**

In the previous chapter I measured *in-situ* the ability of sponge *R. globostellata* living in the lagoon of Bouraké to recycle nutrients and organic matter (Maggioni et al., 2023). Data showed that the massive sponge consumed DOC and produced detritus. However, the process was stopped when sponges underwent very high temperatures (up to 31°C), i.e., the sponge did not produce detritus. While it was possible to obtain these results because the measurements were made *in-situ* and using a sponge species likely adapted to extreme conditions, it was not possible to identify if temperature was the cause of the change in the C and N allocation. Indeed, in experiments conducted in the nature, multi factors can act in combination and synergy. In addition, I only used sponges from Bouraké,
lacking a control population living at “normal” reef conditions. Based on these preliminary findings, in the present chapter i) only the effect of temperature was tested by simulating in aquaria the thermal regime experienced at Bouraké and at a control reef; ii) enriched stable isotopes were used to follow the allocation of C and N into the sponge’s tissue and/or detritus; iii) the responses of sponges living at the extreme reef at Bouraké were compared with a control reef.

Results of this chapter shows that sponges from Bouraké, incorporate more ¹³C and ¹⁵N as coral mucus into the tissue than they release as detritus, and that these rates double at the high temperatures of 32°C. In contrast, the control sponges have an opposite response at elevated temperatures, with less ¹³C and ¹⁵N incorporated in the tissue, and more released as detritus. The ability of sponges to uptake the DOC produced by the reef is well known (e.g., Yahel et al., 2003; Hoer et al., 2018; McMurray et al., 2018; Wooster et al., 2019; Hildebrand et al., 2022), as well as the role of the associated microbes in the C and N cycles (e.g., Hoffmann et al., 2009; Feng and Li, 2019). They can be recycled and released as detritus, mainly in encrusting sponges (sponge loop pathway, De Goeij et al., 2013), and/or retained as biomass, mainly in massive sponges (e.g., Pawlik et al., 2016; McMurray et al., 2018). The factors triggering the two distinct pathways have not been fully clarified. In agreement with my previous *in-situ* measurements (Maggioni et al., 2023), sponges from Bouraké incubated at 28°C recycled DOC and discharged detritus during incubations (i.e., sponge loop pathway) (Figure 4.2). In contrast, sponges from the control reef incubated at the same temperature did not produce detritus, as already found for several massive sponges (e.g., Pawlik et al., 2016; McMurray et al., 2018). These findings were corroborated by ¹³C and ¹⁵N incorporation in the form of coral mucus, and released as detritus. Indeed, at 28°C sponges originated from the control reef showed higher accumulation of ¹³C in the tissue than sponges from Bouraké (average ratio Tissue T:Detritus D = 3.2 vs 1.6, respectively), while the recycling of N was similar between sponge populations (T:D = 2.11 vs 2.07, respectively) (Figure 4.5). These results suggest a different C metabolism that may depend, among other factors, on the environmental characteristics of the sponges’ origin.

Responses were exacerbated at the high temperature showing higher variations in their uptake and incorporation rates. Indeed, at 32°C the majority of sponges, both Bouraké and control, always continued to uptake DOC (Figure 4.2). Most importantly, Bouraké sponges stopped producing detritus, while the control sponges showed high variability with several specimens producing detritus (Figure 4.2). The ¹³C and ¹⁵N incorporation clearly showed that the differences between the two sponges depend on the sites of origin. While the Bouraké sponges incubated at 32°C stored more ¹³C and ¹⁵N in the tissue than in the detritus, when compared to 28°C (T:D = 2.4 at 32°C vs 1.6 at 28°C
for C, and 3.1 vs 2.1 for N), control sponges showed an opposite trend with a tendency to reduce their incorporation in the tissue, and significantly increase incorporation in the detritus (T:D = 1.2 at 32°C vs 3.2 at 28°C for C, and 0.8 vs 2.11 for N) (Figure 4.5). These results suggest that sponges adapted to lower temperature (control) could be stressed when exposed to high temperature of 32°C, and they increase cell turnover to maintain tissue homeostasis, resulting in higher cell shedding and tissue degradation (i.e., higher detritus). This was already confirmed during the recovery period of this experiment, because 8 sponges from the control reef died or showed signs of tissue necrosis, while Bouraké sponges did not show any evident sign of stress. Alteration in choanocyte chambers size and density has already been reported on non-adapted sponges exposed to thermal stress (> 31°C) (Massaro et al., 2012). Under stress, sponges continually shed choanocyte cells which undergo a rapid turnover (De Goeij et al., 2009), and consequently produce detritus (Maldonado, 2016). Unfortunately, our short-term experiment did not enable us to follow the fate of sponges in the long term, and see if this stress at 32°C was followed by tissue necrosis and injury or even the sponge death. However, it has been reported that high temperature of more than 30°C induced mortality in sponges (e.g., López-Legentil et al., 2008; Bell et al., 2018b; Ramsby et al., 2018b). Further long-term experiments in aquaria and a reciprocal transplantation of this species between Bouraké and the control reef will help to shed light on its ability to cope with thermal stress.

The Bouraké sponge seems to better cope with a short-term exposure to high temperatures, likely because this population have already experienced chronic exposures to temperatures around 28°C with peaks at 33°C. For example, in summer 2020 sponges experienced temperature higher than 28°C during 40% of the summer season, and species experienced fluctuations in daily temperature of up to 6.5°C (Maggioni et al., 2021). This hypothesis is in agreement with recent studies suggesting that reef organisms living in fluctuating temperature environments are more resistant to temperature stress (e.g., Oliver and Palumbi, 2011; Rivest et al., 2017). Indeed, as we previously found, the respiration rates of *R. globostellata* from Bouraké was not significantly affected by an increase in temperature between 26°C and 32°C, suggesting a degree of adaptation (Maggioni et al., 2023). In agreement, at the high and fluctuating temperatures tested during this experiment, ¹³C and ¹⁵N were still more incorporated in the tissues than in the detritus, when compared to 28°C. This response involved a tradeoff causing a detrimental effect on the relative availability of C and N for other trophic levels. I believe that *R. globostellata* have developed some adaptive strategies to save energy in the tissue and avoid distress during the most extreme environmental conditions, fostering its growth instead of recirculating nutrients. This hypothesis is in agreement with a recent study on sponge's transcriptome showing that sub-lethal temperature of 32°C may promote tissue growth and protect sponge cells
against extended stress conditions, therefore increasing the sponge ability to mitigate cellular damage (Guzman and Conaco, 2016).

Although we did not measure microbes associated with *R. globostellata*, it is known that the species has a high-microbial abundance (HMA, Moitinho-Silva et al., 2017a), and that associated bacteria play an important role in the sponge metabolism, especially with regard to N cycling (e.g., Taylor et al., 2007). Variable NOₓ production and NH₄⁺ recycling were already measured in *R. globostellata* under different tide conditions (Maggioni et al., 2023). Here, we found that Bouraké sponge at 32°C incorporated more N in the tissues than at 28°C, and likely used it for bacterial metabolic processes that could give them the surplus of energy necessary to support elevated temperatures. Another strategie to cope with high temperatures could be the reduction of the cell turnover, favoring cell proliferation than cell shedding. This could allow sponges to save energy to maintain their health during thermal stress avoiding cells degradation (i.e., detritus production), a protection mechanism that control sponges seem not to have.

One potential factor enabling *R. globostellata* to cope with the thermal stress could be related to its ability to rely on different sources for C, since I found differences in the uptake of food between 28°C and 32°C, and for both Bouraké and control sponges. Food selection may result from either passive processes, in which the physical properties of the sponge filtering lead to differential uptake of picoplankton cell types, or active processes, in which food selection is mediated by sponge behaviour (Jürgens and DeMott, 1995; Maldonado et al., 2010). In general, sponges have been reported to selectively filter phototrophic bacteria over heterotrophic bacteria (Hanson et al., 2009; Topçu et al., 2010; Yahel et al., 2006), but some sponges prefer the relatively rarer but labile phytoplankton when compared to the numerical dominant heterotrophic bacteria (Maldonado et al., 2012; McMurray et al., 2016). *R. globostellata* from Bouraké seems to use passive filtration mechanisms (i.e., not selective) when food is not limiting in the environment (Maggioni et al., 2023). Indeed, *in-situ* incubations showed that the abundance of phytoplankton and bacteria during tides had a great effect on sponge metabolism. Therefore, *R. globostellata* fed more on phytoplankton during the high tide, and inversely, more on detritus and bacteria during the low tide, likely because their respective abundances in seawater differed with the tide. In the present study, blank-normalized ingestion rates measured during incubations in chambers containing the same pool of water (Appendix C - Table C4.2), showed that at 28°C, sponges from Bouraké mostly filtered on bacteria and DOC (Figure 4.3), in the same way of control sponges, and although the latter showed higher percentages. However, Bouraké sponges consumed more phytoplankton and less POC compared to control sponges, suggesting a different metabolic carbon food source selection depending on the sponge origin. At
32°C, we did not find any difference between the two sites, with bacteria as the main carbon sources filtered. This result confirm a previous study showing that temperature above 31°C also caused a behavioral shift in sponges feeding with higher selective filtration efficiency for phototrophic microbes (Massaro et al., 2012). Similarly, the results of this chapter suggest that high temperatures may change the preferred source of C, possibly toward a more energetic and less cost food source. Future works could include NanoSIMS technique to better understand the utilization of the incorporate C and N in sponge tissues.

Although sponges in both sites accumulated carbon derived coral mucus in their tissues, and released it in the form of detritus, the balance between accumulated and excreted C and N changed according to the thermal regimes and the sponges' origin. Such a change in C and N allocation at high temperature, with less detritus being redistributed to the surrounding ecosystem, could be a tradeoff used by the Bouraké sponge population to cope with the extreme environmental conditions (i.e., high temperatures). This chapter highlighted that ocean warming could change the way by which sponges recycle C and N, altering the quality availability of essential sources of energy in coral reefs.
**CHAPTER 5:**
**GENERAL DISCUSSION**
This PhD thesis explored the responses of a tropical sponge to extreme environmental conditions (high temperature, low pH, and low dissolved oxygen) in the Bouraké natural laboratory (New Caledonia). There, the average seawater temperature is similar to those forecasted in the context of global ocean changes, and physico-chemical parameters fluctuate with the tide (Maggioni et al., 2021). The seawater pH and dissolved oxygen decrease during falling tide and reach the lowest and extreme values at low tide (7.2 pH and 1.9 mg O₂ L⁻¹ at Bouraké vs. 7.9 pH and 5.5 mg O₂ L⁻¹ at control reefs). This thesis aims to fill the gaps in our knowledge of how these conditions affect the recycling of sponge nutrients and organic matter. First, I performed a long-term physical, chemical, and benthic characterization of the Bouraké lagoon (chapter 2), which was used after to study the effects of extreme environmental conditions on sponge metabolism.

*Rhabdastrella globostellata* was chosen as model and incubated *in-situ* using benthic chambers during normal (high tide) and extreme (low tide) conditions (chapter 3). Changes in physical and chemical seawater parameters during the incubations were examined and suggested that temperature may be the main factor affecting the carbon cycle of sponges in Bouraké (stop of detritus production). To confirm this hypothesis, an *ex-situ* aquaria experiment was performed to explore the effect of the high temperature on the ability of *R. globostellata* to recycle nitrogen and carbon (chapter 4). The responses of *R. globostellata* from Bouraké and a control site were analyzed at 28°C and 32°C. The ability of the sponge to recycle nitrogen and carbon was assessed by tracking labeled ¹³C and ¹⁵N in both sponge tissue and detritus. Result showed that high temperature drives the shift in C and N allocation in Bouraké sponges.

In this chapter, I will review the main results of my research and show how the responses of Bouraké sponges suggest their ability to cope with extreme conditions. Finally, I will discuss how my results could help to predict sponge responses in future coral reefs and to better understand to what extent their responses will affect the overall reef ecosystem.

**5.1 Rhabdastrella globostellata shows different strategies to cope with extreme environmental conditions**

Sponges influence ecosystem functioning by modifying both biotic and abiotic factors (Bell, 2008). To survive environmental changes, sponges must either acclimate or adapt, like other benthic marine invertebrates (Hoegh-Guldberg, 1999). Acclimatization involves changes in various physiological processes to perform better under new environmental conditions (i.e., acclimatory mechanisms), while adaptation occurs through the selection of individuals better able to cope with the new environmental conditions within the population (Hoegh-Guldberg, 1999).
As described in the third chapter, different environmental conditions (normal and extreme) lead to different responses in the metabolism of *R. globostellata* in Bouraké. The main metabolic difference was found in sponge silicate and carbon cycling. Both silicate and dissolved organic carbon were consumed more during the extreme conditions in the Bouraké lagoon (low tide). During low tide, sponges can benefit from the higher concentrations of orthosilicic acid and organic carbon in the environment to store them as a reserve, and to provide energy and build their skeleton. Some authors have reported that different environmental conditions can lead to changes in the morphology and composition of spicules (e.g., Bell et al., 2002; Meroz-Fine et al., 2005). For example, the sponge *Tetilla* sp. has a higher proportion of spicules in exposed shallow waters than in still water (Meroz-Fine et al., 2005). A higher proportion of spicules results in a stiffer and more robust sponge shape that has minor deformation, even under high water velocity (Palumbi, 1986). A robust skeleton is likely to facilitate the adaptation of sponges to climate change (Bennett et al., 2017). In addition to the number of spicules, their dimensions (thickness and length) contribute to rigidity and flexibility of the sponge. Sponges living in high energy sites have longer and thicker spicules than low energy sites (Bell et al., 2002). Similarly, in the Bouraké lagoon, sponges may accumulate orthosilicic acid to build numerous long and thick spicules in order to maintain skeletal growth, rigidity, and flexibility to cope with the environmental change and the high current velocity that occurs during rising and falling tides (chapter 2).

In coral reef ecosystems, encrusting sponges uptake DOM and release it as detritus (e.g., De Goeij et al., 2013; Alexander et al., 2014; Rix et al., 2018), while several massive sponges prefentially store it as biomass (e.g., Pawlik et al., 2016; McMurray et al., 2018; Pawlik and McMurray, 2020). While the Bouraké *R. globostellata* is a massive sponge, I found that it mainly transformed DOM and released it as detritus (i.e., sponge loop pathway). Specifically, my data suggest that *R. globostellata* in Bouraké preferentially uses DOM to maintain cell turnover and rejuvenation, which keeps the sponge healthy. This different strategy from other massive sponges could be due to the large amount of nutrients and organic matter available in the Bouraké lagoon, which allow the sponge to harness this energy to renew its cells, and perhaps even to grow. This hypothesis was also supported by the *ex-situ* experiment (chapter 4) which highlighted different carbon and nitrogen allocations between *R. globostellata* specimens from Bouraké (i.e., detritus production) and the control site (i.e., less detritus production), both exposed to a ‘normal’ temperature of 28°C. My data show that responses are different if sponges are from different sites and may be related to food availability. This result is consistent with those of Alexander et al. (2014) who demonstrated in aquarium that *Mycale microsigmatosa* growing on mangrove roots in Curaçao (Caribbean island) where food is abundant, had better temperatures tolerance and a higher cell proliferation rate than sponges from reference
reefs. This change in strategy seems to be permanent in sponges from Bouraké (i.e., probably resulting
from an adaptive response) because they have maintained the *in-situ* response (i.e., detritus
production) also when environmental conditions and food availability were different (i.e., in
aquarium), also highlighting possible different ecological traits between the two sponge population.

At higher temperature conditions, the sponges from the control site produced more detritus and showed higher accumulation of ¹³C and ¹⁵N in the detritus, suggesting that the sponges underwent stress, which caused a severe cell degradation. Conversely, *R. globostellata* from Bouraké accumulates more ¹³C and ¹⁵N in the tissues than in the detritus when the temperature exceeds 31°C, suggesting that the sponge has developed strategies to resist extreme conditions. These differences in strategy could be related to sponge thermal history. Sponges living in fluctuating temperature environments could be more tolerant to temperature stresses (e.g., Oliver and Palumbi, 2011; Rivest et al., 2017). Bouraké lagoon frequently exceeds 31°C in summers, and if we assume this has occurred for centuries, this specific metabolism could be the result of an adaptive response of *R. globostellata* to recurring thermal stress to maintain their state of health. In this case, carbon and nitrogen are accumulated more in the sponge tissues at 32°C than at 28°C. Although it is clear that sponges store carbon and nitrogen in their tissues, I have not identified the mechanism used for this. Carbon and nitrogen stored in sponge tissues are probably used in different metabolic processes and provide additional energy for sponge health and growth. This change in energy allocation can be the result of an adaptive mechanism that allows *R. globostellata* to perform better under stressful temperatures and, thus, thrive in the Bouraké lagoon **(Figure 5.1)**.

During the extreme conditions in 2020 (i.e., when the change in strategy was observed *in-situ*), *R. globostellata* took up more amount of carbon energy available for metabolic processes. An approximation of the carbon balance in sponges could be defined using the I:R ratio where I is the carbon ingested (DOC + POC) and R is the carbon used for respiration. Since the sponge showed no significant difference in respiration rates between the two environmental conditions (normal and extreme), we can assume that the sponge does not need to use more carbon energy for respiration to withstand extreme conditions (chapter 3). Respiration rates in sponges are considered a proxy for understanding sponge fitness and stress response in organisms (Coma et al., 2002). My results suggest that there is no stress response in *R. globostellata* under extreme conditions, which means that the metabolic physiology of the sponges allows them to respond to environmental changes (e.g., Coma et al., 2002; Morley et al., 2016; Bell et al., 2017). Higher respiration rates generally means that more energy is needed to maintain the sponge's basal metabolism and life processes (Clarke, 1991). The high availability of food in the Bouraké lagoon (chapter 2) may help the sponges to maintain their
basal metabolic rate and enhance carbon storage under stressful conditions. *R. globostellata* may then
compensate for metabolic losses (including higher respiration rates) and ensure an optimal allocation
of excess energy for metabolic processes such as growth, as reported for other organisms (Kozłowski,
1992; Perrin and Sibly, 1993). This hypothesis is supported by other studies showing that when food
is not limiting, high growth rates and biomass accumulation can be supported even under stressful
conditions (Sokolova et al., 2012). These responses could allow the sponge to keep their basal
metabolism and thrive well also under the extreme condition in Bouraké, suggesting a possible level
of adaptation of the sponge to these conditions. This hypothesis is reinforced by the fact that *R.
globostellata* increased its respiration rate following the temperature increase between 2018 and 2020
(+4°C) (regardless of the condition) without affecting its survival. It seems able to reach a new basal
metabolic "equilibrium" under changing environmental conditions.

<figure><img src="image_41.png" /><figcaption>**Figure 5.1.** Synthetic scheme of the main strategies used by the Bouraké sponge *R. globostellata* to cope (i.e., adaptive response) with a change in temperature.</figcaption></figure>
The higher accumulation of carbon and nitrogen in *R. globostellata* from Bouraké also suggests a greater exchange of carbon and nitrogen between the host cells and the symbiont bacteria. Microbes associated with sponges are assumed to play an important role in mediating the sponge uptake of DOM (Weisz et al., 2008). The sponge symbionts may play an important role in its adaptation. Recent studies showed that the microbiome of OA-tolerant sponge species had increased their functional potential for energy-efficient carbon and nitrogen metabolism (Botté et al., 2019), contributing to the ecological adaptability and plasticity of the holobiont (Bang et al., 2018). Similarly, the bacterial community associated with *R. globostellata* could contribute to carbon and nitrogen assimilation (extra energy) and could play an important role in their survival and adaptation mechanisms to the extreme environmental conditions of Bouraké. Although this bacterial contribution needs to be further demonstrated, in my thesis carbon and nitrogen seem to be mainly stored in the sponge biomass at 32°C, suggesting a major involvement of symbionts at high temperatures. Among the associated microbial community, the photosynthetic microbes could have different response to OA and OW, ranging from no impact (Bell et al., 2022) to a decrease in both steady state and maximum quantum yields of their PSII (photosystem II) fluorescence (e.g., Lesser et al., 2016). By measuring the photosynthetic activity in *R. globostellata* under normal and extreme conditions, my results revealed that photosymbionts were negatively affected (i.e., less oxygen production and reduction in photosynthetic activity) by extreme conditions, suggesting that they may have little contribution in the sponge acclimation responses to extreme conditions. Heterotrophic sponges have been reported to be more tolerant to temperature anomalies than phototrophic sponges (Bennett, 2017). The studied species *R. globostellata* resulted mainly heterotrophic (P:R ratios < 1.5, following Wilkinson, 1983), suggesting that the heterotrophic processes, and the heterotrophic symbionts, may play an important role in the sponge adaptation to extreme conditions. Similarly, recent studies have shown that an increase in heterotrophic bacterial abundance can promote greater sponge productivity (e.g., Perea-Blázquez et al., 2012) and that eutrophic conditions could make bio-eroding sponges less dependent on their photosymbionts (Webb et al., 2017).

My work showed that sponges have metabolic plasticity to cope with different environmental conditions. Although I have only studied the responses of one sponge species at the local spatial level, this study demonstrates that *R. globostellata* can cope with extreme conditions in an advantageous way. In summary, the success and adaptation of *R. globostellata* in Bouraké, despite the extreme physico-chemical environmental conditions, could be linked to i) the fluctuating environment, which provides the sponges with regular phases of extreme conditions and optimal conditions, likely allowing them to recover from stress, and ii) the wide range of heterotrophic inputs, nutrients, carbon, and nitrogen sources that can help sponges cope with extreme conditions through additional energy.
If we consider that sponges living in the Bouraké lagoon have been exposed to these environmental conditions for decades, more likely centuries, these responses could be the result of adaptive mechanism take in place by the sponge to thrive under extreme conditions such as high temperature.

## 5.2 Bouraké sponges: a model of adaptation for future coral reefs?

My results show that *R. globostellata* has developed strategies to survive and thrive in the extreme environment of Bourake. However, it is questionable whether these results are appropriate for predicting how organisms will respond to future conditions elsewhere.

Although we cannot predict whether the natural fluctuations expected for other coastal habitats will be comparable to what has been measured in the Bouraké lagoon, the models predict larger fluctuations in physico-chemical water parameters. Seasonal pH amplitude could increase by 80%, extreme values of ocean acidity will be more frequent, and pH variability will increase further at sites where variability is already high today (Kwiatkowski and Orr, 2018; Burger et al., 2020). The situation could be similar also for seawater temperature. At Bouraké, the mean values of the environmental parameters (pH~T~ = -0.3 units, DO = -26%, and T = +3°C, in contrast to current values) nevertheless approach the values predicted by the models under the IPCC scenario (pH~T~ = -0.3 units, DO = -4%, and T = +3°C), suggesting that this site can be used to study the adaptive responses of organisms to future conditions.

In contrast, at Bouraké, changes in physical parameters are driven by the tide, exposing organisms to normal or extreme conditions every *ca.* 6 h. The response mechanisms observed in this study were thus the result of this fluctuation, which of course allow the sponge to limited periods of quasi-normal conditions. Over time, the tide could have exposed the sponges to the stressful conditions long enough to induce adaptation but short enough to avoid sponge mortality as reported in some coral studies (e.g., Oliver and Palumbi, 2011; Castillo et al., 2012; Palumbi et al., 2014; DeCarlo et al., 2019). Although this could be true for temperatures, pH and DO inside the Bouraké lagoon rarely reach normal values exposing organisms to these extreme values for decades, even centuries, which has probably allow long-term selection of resistant organisms and resulted in local adaptation. It is questionable whether the same mechanisms will be able to take place in different environments where organisms are likely to be exposed to stressful conditions more rapidly and more regularly. However, recent studies have shown that rapid adaptation and plasticity in certain physiological traits may be heritable, helping organisms (e.g., corals) to cope with rapid environmental change as well (e.g., Torda et al., 2017; Putnam, 2021).
Overall, the interpretation of my results about future climate change responses should be taken carefully, as several other environmental factors, besides physical parameters, such as nutrients and organic matter concentrations, varied at the site and are likely to influence the organisms' adaptive responses. Coastal lagoons, such as Bouraké, are highly productive ecosystems that serve as nursery grounds for many marine species (Anthony et al., 2009) and receive terrigenous runoffs from rainfall, sewage, and/or agricultural fertilizer (Taylor et al., 1995). In Bouraké, this organic matter input is amplified by the surrounding mangrove, considered one of the world's most productive ecosystems (e.g., Alongi et al., 2004; Dittmar et al., 2006; Bouillon et al., 2008). The benthic communities of Bouraké have access to a wide range of heterotrophic inputs, nutrients, carbon, and nitrogen sources. Although these inputs can become toxic if they are too concentrated, they could also help organisms cope with extreme conditions.

No natural laboratory is perfect for studying the impact of future climatic conditions, but the lagoon of Bouraké offers a realistic combination of multiple factors that will affect future coral reefs. Despite the small spatial scale offered by the Bouraké natural laboratory, my results provide essential insights into the potential mechanisms of sponge acclimation and adaptation to different environmental conditions. The temperature in coral reefs is expected to increase by 3°C in the future, a condition met in the lagoon of Bouraké during low tide. My results experimentally demonstrate that *i*) above a certain threshold (> 31°C tested here) temperature induced a change in organic matter allocation in *R. globostellata*; and *ii*) the observed responses were unique to Bouraké sponges, probably owing to their presumed ability to cope with extreme temperature variations (Figure 5.1). This suggests that while the Bouraké sponges could maintain their normal metabolic and allocate more resources to the tissue, the control sponges probably experienced metabolic stress and cell degradation.

Assuming that contemporary sponges will adapt as they did sponges in Bouraké, this study suggests that future coral reefs are likely to be dominated by larger sponges that invest more energy in their individual growth. The change in C and N allocation may have consequences in the amount of detritus available for the other trophic levels in the ecosystem. Less detritus will be incorporated by sponges-associated detritivores, and less detritus will reach different trophic levels. Carbon and nitrogen are mainly stored in the sponge tissues. Sponge predators would be advantaged because a high sponge biomass would be available for them. However, only a limited number of species of fish, turtles, and nudibranch species are able to cope with sponge secondary metabolites in their diet. Consequently, I believe that the potential use of sponge biomass by predators remains a (very) minor organic matter pathway. The biomass accumulated by sponges may re-enter the system when the temperature conditions change (i.e., detritus production) or when the sponge dies, thus following a
necrophagous/detritivorous trophic pathway. Although adaptation of all reef organisms and their interactions will need to be considered and that these results remains hypothetical and restricted to a specific case, my work has highlighted that ocean warming could change how sponges recycle C and N, and thus the food quality and availability in the reef.

Several personal observations noticed that the reefs in Bouraké have changed over the past years, with significant negative impacts on corals (i.e., bleaching and mortality), while the sponge abundance and their massiveness remain high. Stressful conditions (rainfall, cyclones, La Niña/El Niño events and temperature rise) appear to have more effect on other organisms than on sponges, which could have less competition for space and continue to take full advantage of high nutrient levels for growth under non-optimal conditions. These observations agree with my results showing that *R. globostellata* could exhibits adaptive mechanisms that allow it to compete successfully in this extreme environment.

In conclusion, this research has highlighted the importance of studying how temperatures can affect the ecological role of sponges in coral reefs, as temperature may be one of the major climate change consequences driving changes in the metabolism of most sponge species. Changes in sponge metabolism, such as different allocation strategies of C and N between sponge tissue and detritus, as well as the ability of sponges to recycle organic matter, could have significant effects on the entire reef. Effects on trophic interactions could lead to a shift towards communities dominated by sponges and other organisms that can adapt to and take advantage by high temperatures.

## 5.3 Future perspectives

Sponges play an important ecological role by recycling carbon and nitrogen in coral reef ecosystems (e.g., Maldonado et al., 2012; De Goeij et al., 2013). Both pathways of carbon recycling in sponges (sponge loop and biomass storage) need to be fully assessed to get a better idea of the carbon cycle (and organic matter in general) in massive and encrusting sponges, as well as the contribution of microbial symbionts in these processes. More than 8,500 sponge species have been described (van Soest et al., 2012), and there is no information on carbon and nutrient cycles for most of them. Although I studied only *R. globostellata*, my research provides an initial and original insight into understanding the effects of extreme conditions, mainly OW, on the sponge nutrient and carbon cycles. Further work should consider more sponge species, both encrusting and massive, and their nutrient and carbon cycles must be assessed under various environmental conditions, including extremes ones. Future studies should prioritize cross-transplanting experiments using other natural laboratories to explore how sponge's nutrient and carbon cycles could change under future conditions.
*Ex-situ* experiments maintaining sponges at elevated temperatures for more extended periods should be performed to understand the level of sponge temperature tolerance and provide better insight into how the sponge carbon and nitrogen cycles might be affected under future temperatures.

While I did not investigate which metabolic processes the sponges used to accumulate carbon, my research showed that under extreme conditions (T > 31°C) carbon is mainly stored in the sponge tissues rather than released as detritus. Future studies using specific techniques to label and follow carbon transfer in sponges (e.g., NanoSIMS) will help to better understand the processes that occur when carbon and nitrogen are no longer released as detritus. Such studies can shed light on the strategies used by sponges to adapt to high temperatures. This innovative approach, should anyway be coupled with classic sponge biomass measurements (i.e., buoyant), pumping rates, and food quality.

Finally, because sponges are holobionts, the responses of the associated microbial community must be considered. Some studies have indicated that changes in the sponge microbiome could be the key to sponge adaptation to new environmental conditions (e.g., Webster and Reusch, 2017; Pita et al., 2018). The role of the microbiome on sponge organic matter recycling still needs to be better understood, particularly its contribution to adaptation under extreme conditions, including OW. I showed several evidence of adaptation in my research, but complementary analyses are needed to demonstrate it fully. Future studies on lipidic contents, heat shock proteins, and transcriptomic could be key to understand if the responses of *R. globostellata* from Bouraké are the result of adaptation or a physiological response under stressed conditions.

**5.4 General conclusion**

Environmental changes, as expected under future climate scenarios, could strongly affect marine species in the ocean. Sponges have different and species-specific responses to climate change and environmental variations. Studies so far have mainly focused on the effects of climate change on sponge physiological traits rather than their ecological role, such as carbon and nitrogen cycles in coral reefs.

My thesis provided 1) the first long-term physico-chemical characterization and assessment of the Bouraké Lagoon benthic community, which is now considered a natural laboratory for studying the adaptation of organisms to extreme conditions; 2) the first insights into the effects of extreme conditions on *R. globostellata* metabolism, showing a significant change in the carbon cycle to cope and thrive in extreme environments; 3) information about the effects of temperature on *R. globostellata* carbon and nitrogen recycling, showing that sponges may be able to tolerate short-term
high temperature exposure (+3°C), and that sponges subjected to high temperatures for decades, even centuries (Bouraké), showed an adaptive response that involve a greater accumulation of carbon and nitrogen in the tissues than in the detritus. Whether these are mechanisms that other common sponges could use to cope with climate change in the future remains to be understood.

In a world where climate change is accelerating, and many of the effects of these changes have yet to be studied on organisms, my thesis provides an example of how a tropical sponge can cope with extreme acidification, warming, and deoxygenation conditions. I showed that the studied sponge could adapt to different environmental conditions taking maximum advantage for their health and suggesting that survival of sponges, and hopefully other taxa, to future environmental conditions, may be possible. However, the possible adaptation to high temperatures may result in a change in metabolic responses and thus a change in trophic interactions in coral reefs.
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