INTERNSHIP REPORT

Impact of the nudibranch Doris verrucosa predation on the ​ ​ sponge Hymeniacidon perlevis community dynamics ​ ​

By Clémence Potel Supervised by María López-Acosta ​ Sept.-Dec. 2020 • Acknowledgement

I would like to thank María López-Acosta who was a wonderful internship tutor. She let ​ me work alongside her in this project, made me discover the world of a marine biologist researcher and gave me great advice for the writing of this report. I would also like to thank Aude Leynaert (and Marguerite Baradat) without whom I would not have been able to do this internship neither had this lovely experience; the divers Isabelle Bihannic, Erwan Amice and Thierry Le Bec for their sympathy and letting me sail with them; and the whole LEMAR institution for the warm welcome. • Abstract (English)

Sponges are one of the most ancestral multicellular animals still living on earth but yet, they are still poorly studied and understood by the research community compared to other marine benthic metazoans. Sponges are known to play lots of essential roles in marine ecosystems such as stabilizing marine substrate and provide microhabitats for smaller benthic organisms as well as they are also involved in several nutrient cycles. Therefore, a better understanding of the dynamics of sponge populations could prevent potential future ecological crises. This study focuses on the impact of the nudibranch Doris verrucosa predation on the sponge Hymeniacidon ​ ​ perlevis in a shallow-water ecosystem in which they cohabit, the maerl beds of the Bay of Brest, France. The predation rate is estimated by the quantification of the sponge volume eaten per nudibranch volume in a day (mL sponge mL⁻¹ nudibranch D⁻¹). To do so, the sponge volume was measured by association of various geometric shapes, before and after being offered to a nudibranch for 24h. The nudibranch volume was also measured after the experience. The predation rate was then calculated by subtracting the final sponge volume to the initial one, and normalized by the nudibranch volume. The experiment has been led 5 times on 30 nudibranchs in the laboratory to investigate intra- and inter-individual variabilities and once on 10 nudibranchs in situ, to measure the predation rate in their natural habitat. From the laboratory experiments, we learnt that there was no specific intra-individual variability and that the main inter-individual activity was due to the nudibranch size. From the in situ incubations, we estimated that the average predation rate is 0.156 ± 0.091 mL sponge mL⁻¹ nudibranch D⁻¹. Then, the predation activity was extrapolated to the nudibranch population in its natural habitat from June to October (the months in which these predators are active) 2020, we found that 41.24 ± 25.12% of H. perlevis biomass is eaten by its main predator, D. verrucosa, ​ in the maerl bed of the Bay of Brest. Therefore, D. verrucosa strongly impacts the H. ​ ​ ​ perlevis population dynamics and its distribution over the year cycle. ​

• Keywords (english): sponge, nudibranch, predator-to-prey relationship, predation ​ rate, sponge population dynamics, marine benthic ecosystems

• Abstract (Français)

Les éponges font partie des animaux multicellulaires les plus ancestraux vivant encore aujourd’hui sur Terre et pourtant, elles sont peu étudiées et peu comprises par la communauté des chercheurs, contrairement à d’autres métazoaires marins benthiques. Les éponges sont connues pour jouer plusieurs rôles essentiels dans les écosystèmes marins comme la stabilisation des substrats marins mais représentent aussi des microhabitats pour les plus petits organismes benthiques et sont impliquées dans les cycles de nombreux éléments chimiques. Par conséquent, mieux comprendre les dynamiques des populations des éponges pourrait éviter de potentielles crises écologiques futures. Cette étude se focalise sur l’impact de la prédation du nudibranche Doris verrucosa sur l’éponge Hymeniacidon perlevis dans leur écosystème naturel, les ​ ​ ​ lits de maërl en eaux peu profondes de la Rade de Brest, France. Le taux de prédation est estimé par la quantification du volume d’éponge mangé par volume de nudibranche et par jour (mL éponge mL⁻¹ nudibranche J⁻¹). Pour y parvenir, le volume d’éponge fut mesuré deux fois par association de différentes formes géométriques: avant et après qu’elle fût offerte au nudibranche pendant 24h. Concernant le nudibranche, son volume fût mesuré à la fin de l'expérience suivant la même technique. Le taux de prédation fût calculé en soustrayant le volume final de l'éponge à son volume initial, et divisé par le volume du nudibranche. Cette expérience a été menée en laboratoire 5 fois sur 30 nudibranches, et une fois in situ sur 10 nudibranches afin de calculer le taux de prédation dans l’habitat naturel des organismes d’étude. À partir des expériences menées en laboratoire, nous avons découvert qu’il n’y a pas de variabilité intra-individuelle particulière mais que la principale variabilité inter-individuelle est due à la taille du nudibranche. À partir des expériences in situ, nous avons estimé un taux de prédation moyen de 0.156 ± 0.091 mL éponge mL⁻¹ nudibranche J⁻¹. Ensuite nous avons extrapolé notre résultat sur la population des nudibranches dans habitat naturel entre Juin et Octobre (ce qui représente la saison d’activité des nudibranches) 2020 et avons trouvé que 41.24 ± 25.12% de la biomasse de H. perlevis a été mangé par son ​ ​ principal prédateur D. verrucosa dans le lit de maërl de la Rade de Brest. Par ​ ​ conséquent, D. verrucosa impacte fortement les dynamiques et la distribution de la ​ ​ population de H. perlevis. ​ ​

• Keywords (français): éponge, nudibranche, relation proie-prédateur, taux de ​ prédation, dynamiques de population des éponges, écosystèmes marins benthiques

• Graphical abstract

• Introduction

Marine sponges are conspicuous animals in benthic ecosystems. Their large spectrum of species make them ubiquitous in marine environments, regardless of latitude and depth, forming in some cases enormous aggregations that may extend over large areas [1]. Sponges play several functional roles in the global marine ecosystem ​ ​ such as substrate stabilization or microhabitats for other benthic organisms. They are also involved in several nutrient cycles essentially silicon, carbon and nitrogen [2,3]. ​ ​ Thus, understanding the dynamics of sponge populations is important to acknowledge the global marine ecosystem functioning and to prevent potential risks to which marine ecosystems could be exposed.

Sponges are known to be poorly predated [4,5]. This feature is due to their ​ ​ mineral skeleton. Most sponge species build their skeletons with mineral material, either carbonate calcium or amorphous silica. Some sponge species combine their mineral skeletons with organic compounds, which are mainly made by spongin, a collagenous-like protein [6]. The whole skeleton causes irritation of their predators’ ​ ​ mouth and gut and the spongin protein is known to be very indigestible for most marine animals. Additionally, certain species of sponges produce chemical toxins in order to ​ deter predators from feeding [7]. The few main sponge predators which have been ​ ​ ​ reported are sea stars, angelfishes, trunkfishes, file fishes and nudibranchs [4,5,7]. To ​ ​ date, most of the studies on sponge predation have been conducted in tropical latitudes, particularly in the Caribbean coral and sponge reefs [4,5,7,8], with only some studies in ​ ​ high latitude environments [9]. Surprisingly, there are hardly any studies on sponge ​ ​ predation in temperate latitudes [10]. This represents a lack of information concerning ​ ​ the sponge communities, given that not only their diversity and role but also the benthic population dynamics highly change across latitudes [11,12]. Here we have studied ​ ​ nudibranchs’ predation on sponges in the Bay of Brest, France (NW Atlantic).

Nudibranchs, also called “sea slugs”, are shell-less invertebrate molluscs. Depending on the species or the family, they feed on a specific kind of prey [13]. For ​ ​ instance, some species, such as those belonging to the Family Dorididae, are spongivores [14]. Along evolution, they have become unaffected to sponges’ secondary ​ ​ metabolites. Two main types of spongivorous exist: the specialists, which only feed on one specific sponge species, therefore they are adapted to predation on this species and are totally unaffected by its defense system. For instance, a correlation between certain nudibranchs’ tooth concavity and certain sponges’ spicules shows a specific adaptation for predation [15]. The second type of spongivores are generalists which ​ ​ feed on several sponges species. Either because they are looking for different sorts of nutrients, either to limit recurrent damages caused by one specific defense mechanism, or simply because they are not affected by any of the sponges’ secondary metabolites

[4]. The​ nudibranch of interest in our study, Doris verrucosa belongs to the family ​ ​ Dorididae and follows the last case scenario: it has mainly been observed feeding on ​ Hymeniacidon perlevis (formerly called Hymeniacidon sanguinea) but sometimes on ​ ​ other sponge species [16]. ​ ​ In this study we focus on the predator-to-prey relationship between the nudibranch D. verrucosa and the sponge H. perlevis by determining the predation rate ​ ​ (i.e., quantity and velocity of sponge biomass eaten by nudibranchs) and consequently, the impact that the nudibranchs have, through predation, on the sponge population dynamics of the marine temperate coastal ecosystem, the Bay of Brest (France). As I have always been intrigued by marine biology but never had the chance to feed my curiosity, this internship was an ideal approach for me to discover this new field of study.

• Context/background

A group of researchers among the Laboratoire des sciences de l’environnement ​ marin (LEMAR), a research unit of the UBO, CNRS, IRD and Ifremer which is located in Plouzané (France), works on the understanding of the marine silicon cycle. My internship tutor, María López-Acosta, focuses most of her work in understanding the ​ ​ role that siliceous sponges play in the silicon cycle of marine environments, and she recently has contributed to identify and quantify the participation of these organisms to the silicon cycle both at regional and global scale [17,18]. In the ocean, the silicon cycle ​ ​ is under biological control. To date, most of the biological processes controlling the cycling of this nutrient has been focused on diatoms’ biology, with other groups of silicifiers considering to play secondary or negligible roles. Nevertheless, recent discoveries highlighted the fact that some silicifiers such as siliceous sponges [18] and ​ radiolarians [19] also​ play an important role in the balance of the dissolved silicon in the ​ global oceans, which can be particularly relevant at regional levels.

The marin silicon cycle is composed of inputs and outputs. The main inputs come from terrestrial silicon which is dissolved by weathering and transported by rivers into the oceans, but also through groundwater, hydrothermal vents and ice melting. For decades, the only known silicon output was through the burial of the diatoms’ cell walls named frustules. This noticeable unbalance between silicon inputs and outputs has triggered the attention of several researchers who decided to lead further research on silicon outputs. Among other investigations, Maldonado and coauthors have recently quantified that sponges represent a major silicon sink at global scale through the burial of their siliceous skeleton. Siliceous sponges absorb dissolved silicon and transform it into biogenic silica ( SiO2 ) to build their spicules, the siliceous pieces that compose their skeleton. At their death, the skeleton ends up buried and silicon returns to its original form: sediments. Every year, sponges participate to the silicon outputs by burying 1.71±1.61 Tmol of Si yr⁻¹ [18]. ​ ​ Considering that sponges are ubiquitous regardless latitudes or depths, represent an enormous benthic biomass and play lots of essential roles in marine environments, major consequences on the global marine ecosystem could occur if their dynamics get significantly impacted. Additionally, there are some basic ecological knowledges regarding sponge population dynamics that are still poorly investigated. Hence, Maria wanted to lead further research on sponge ecology and more specifically on how nudibranchs impact the sponges community dynamics by their predation. In June 2020, three months before the beginning of my internship, the amount, volume and activity of D. verrucosa and H. perlevis have been reported in their natural habitat, ​ ​ the maerl beds of the Bay of Brest.

The nudibranch D. verrucosa is often observed on H. perlevis. In order to make ​ ​ ​ sure that this sponge species is the main source of feeding of D. verrucosa, we ​ ​ observed, after the sampling, some of their fecal pellets with an optical microscope to determine the type of sponge consumed. They were full of megascalere, near to rounded ends spicules (“needle spicules”) which are specific to Hymeniacidon [20]. We ​ ​ ​ can conclude that, in their natural habitat at the Bay of Brest, D. verrucosa mainly feeds on H. perlevis. Our study on the predator-prey relationship between those species is ​ ​ complementary to the natural alimentary habits of D. verrucosa. Another interesting ​ point can be highlighted from this fecal pellets observation: the siliceous spicules are found intact which means that D. verrucosa does not feed on sponges for their silicon ​ content but for their organic compounds.

• Material and Methods

1) Study site

The Bay of Brest, located in Brittany, France, is a temperate shallow-water embayment. This ecosystem has various types of benthic habitats such as shallow muds, rocky as well as maerl beds, in which a rich and abundant benthic community ​ lives. All the organisms were sampled in a maerl bed called Lomergat located on the southern side of the Bay (48°17.197'N; 4°21.279'W) (Fig.1). The average surface of ​ ​ Lomergat was estimated with google maps as 3810000 ± 100 m². The organisms used ​ ​ for conducting the laboratory experiments were collected during September and October 2020 by SCUBA (self-contained underwater breathing apparatus) diving.

2) Laboratory experiments

Laboratory experiments were conducted in 2 sets of 15 nudibranchs and 5 times each.

The experiments were led on D. verrucosa separately. Hence, in order to prevent ​ them from meeting during the experiments, 18 cubic cages were built (25x25 cm) with basic plastic garden fences recovered by a small mesh net by stitching. We added plastic tubes along the length, width and height to maintain the fence straight. Cage building was very cautious because nudibranchs are able to change their shape and size into extremely small circumferences (about 0.5 cm). Only plastic was used to prevent any unwanted rust which could contaminate the sea water and kill the sponges. In fact, sponges are filter-feeders which means that they are extremely sensitive to water quality. Thus, seawater was continuously incoming and outcoming the aquaria in order to have constant clean water and to simulate a natural water flow. In addition, no chemical contaminations must occur: hand cream, hydroalcoholic gel or even soap can be lethal for sponges. We made sure to wash our hands under running seawater before any manual manipulations of the organisms.

During laboratory experiments, each D. verrucosa was isolated in a cage, where ​ ​ they received a dedicated H. perlevis for 24 hours. For each experiment, 18 sponges ​ ​ were used: 15 were given to nudibranchs and the 3 remaining sponges were used as controls, which means that they were not attributed to any nudibranch. Sponges were placed in the center of their cage and not purposely in front of their predator. To determine the quantity of sponge eaten by one nudibranch per day, sponges’ volumes were measured before and after the experiment. Their volumes were measured by association of various geometric shapes which were analogous to the sponge’s shape. Volume measurement by water displacement, which is more precise, cannot be used on living H. perlevis. Unlike other marine benthic organisms, most sponges die when they ​ ​ are exposed to air. Volume measurement by approximation to geometric shapes is the most commonly used method for sponges [21,22]. The geometric shapes that were ​ ​ mainly associated with the sponge shape were: parallelepipeds, truncated cones, cones, cylinders and ellipses. All of them were estimated by measuring the linear parameters (length, width, height, diameters) with a ruler while the sponge was placed in an aquarium with transparent walls. Sponges were therefore never exposed to air along the experiments. Measurements were taken at each side of the sponge to capture the anomalies that sponges showed, as they were not always completely symmetrical. We used the mean value of each linear measurement for volume calculation. Measurements were always taken by the same person. The precision of linear measurements was 0.1 cm.

At the end of the experiment, after removing the sponges from the nudibranchs cages, the volume of each D. verrucosa was measured following the same method as ​ for sponges, but this time by approximating their volume to a half elliptic-cylinder shape.

From one experiment to another, nudibranchs were starved approximately 48h and sponges renewed. This fasting period is necessary to normalize the conditions of the animals for each experiment and is often used in predation studies [23]. ​ ​ Finally, at the end of each set of experiments, once the nudibranchs were not experimentally used anymore, we measured the wet weight of each nudibranch, making sure to absorb the excess of water with a towel. Biomass data of nudibranchs were ​ tested with regression analysis. The obtained equation permits to estimate the wet weight from the volume data and vice-versa (see Annex 1). ​ ​ ​ ​ To observe nudibranch activity during laboratory experiments, some nudibranchs were recorded during 1 hour with a GoPro hero 8 black at the beginning and at the end of each experiment by time lapse mode with an interval between pictures of 1min. The time lapses can be found on the supplementary materials (see Annex 2.). ​ ​ ​

Predation rate of D. verrucosa on H. perlevis was calculated as the difference of ​ sponge volume at the beginning and at the end of each experiment and normalized by time and nudibranchs biomass (mL sponge D-1 mL-1 nudibranch). The maximal error is ​ ​ ± 10% of the initial sponge volume due to measurement precision. It was estimated thanks to the control sponge measurement differences between the beginning and the end of the experiment.

3) In situ experiments

The in situ experiment was conducted on 1 set of 10 nudibranchs at the study site Lomergat, which is the natural habitat of the organisms. In this case, 13 H. perlevis ​ were used: 10 were given to D. verrucosa and 3 were used as controls. ​ ​ For the in situ experiment, cylinder cages of 20cm height and 25cm in diameter were built following the same cages structure than Leber [24] and using the same ​ materials as for laboratory experiment’s cages. They were fixed on the sea floor with concrete iron. (Fig.2 and Fig.3) ​ ​ ​ ​ The experiment procedure was the same as the laboratory’s. The organisms were sampled the same day and from the same place where the in situ experiment was led. Because the in situ site is not easily accessible, the 24h experience duration was more flexible (± 30min) than at the laboratory. As for the laboratory experiments, sponge and nudibranch volume was measured with a ruler before and after the experiment. Nudibranch volumes were also measured in the laboratory after the experiment, as well as their wet weight. Predation rate of D. verrucosa on H. perlevis ​ was calculated as in the laboratory experiments, that is, as the difference of sponge volume at the beginning and at the end of each experiment and normalized nudibranchs -1 -1 biomass and by time (mL sponge mL ​ nudibranch D ).​ ​ ​ Time lapse videos were made of the 3 first hours of the experiment following the same mode that in the laboratory (see Annex 2.). ​ ​

4) Extrapolation of the predation activity to the natural habitat

To extrapolate our experiment results to the natural environment of H. perlevis ​ and D.verrucosa, we first determined their natural distribution, that is the average ​ ​ biomass and abundance in the study site (Lomergat maerl bed).

Sponge biomass was monitored with a 1x1 m quadrat that was randomly positioned on the seafloor. It is important to randomly throw the quadrat in order to have more reliable measures and therefore an accurate abundance estimation of reality. All the H. perlevis within the random 1x1 m quadrats were counted and their volumes ​ measured with a ruler as it is explained above (see section “Laboratory experiment”). We measured the sponge abundance and biomass in the natural habitat twice along 2020: in June (n=22) and in November (n=12). The abundance of nudibranchs was determined by 25 m transects which were also positioned in a random spot within the study area. At each meter square along the line transect, nudibranchs were quantified, their length measured in situ with a ruler and their activity registered (i.e., if they were eating or not). Nudibranchs were also measured twice in 2020 (June, n=6; November, n=13). From May to October, D. verrucosa are abundant and actively feed in the Bay; ​ ​ during the other months they are scarce and non-active. Therefore, we monitored the ​ nudibranchs in June to know their number during the season while they are abundant and actively feeding on H. perlevis, and in November to know their abundance at the ​ ​ end of the season.

A relevant correlation (n=40, R²=0.9257, p-value<2.2e-16) between nudibranchs length and volume was established thanks to the experimental nudibranchs’ measurement data. From this equation (volume=1.80448 × length; see Results) we were ​ able to estimate the volume of each nudibranch along the transects. Other correlations such as the nudibranch volume depending on the width and height can be found in the supplementary material (see Annex 3. and Annex 4.). Finally, D. verrucosa and H. ​ ​ ​ ​ ​ ​ perlevis abundance and biomass were normalized to m2, as well as the predation ​ ​ activity of D. verrucosa on H. perlevis. ​ ​ ​ ​

Before using our experimental data for the extrapolation, we compared the predation rate of the laboratory and the in situ experiments to determine if they belonged to the same population and therefore work on the two data sets altogether.

The t-test is the most commonly used test for a mean comparison between two groups. Yet, it can only be applied if the following conditions are verified: the populations must each follow a normal distribution and have common variances. Those conditions can be verified, respectively, with the Shapiro and f-test tests. The Shapiro test revealed that the predation rates from both experiments follow a normal distribution, with a p-value=0.1442 (i.e.; p-value>0.05) for the laboratory and p-value=0.45 (i.e.; p-value>0.05) for the in situ. As for the f-test, we obtain a p-value=0.406 (i.e.; p-value>0.05) which means that the variances are significantly similar. Considering that both conditions were verified, we used the t-test and obtained a p-value=0.003 (i.e.; p-value<0.05), which means that the predation rates measured in the laboratory and in situ were significantly different. In other terms, the laboratory and in situ experiments could not be used as the same set of data, but two different ones. The laboratory data set included the 2 sets of nudibranchs altogether because their predation rates were significantly similar (p-value=0.8326>0.05 by t-test). As it is illustrated in the figure 7 (Fig.7), the in situ predation rates are significantly lower than the one from the ​ ​ laboratory. This dissimilarity can be explained by the 48h fasting period that we imposed to the nudibranchs between each laboratory experiment. On the contrary, for the in situ experiments, nudibranchs were directly collected from their natural environment and immediately put in the cages with a sponge. Thus, their predation activity from the last couple of days was unknown. Therefore, the data from the in situ experiments were used for the extrapolation since its conditions are closer to reality and lead to a more conservative approach. As for the data from the laboratory experiment, they were used to determine the intra-individual and inter-individual variabilities among predation activity.

The average biomass of H. perlevis (mL) eaten by D. verrucosa in the maerl bed ​ ​ ​ ​ of Lomergat from May to October was calculated by multiplying the following figures: in ​ ​ situ mean predation rate (mL sponge mL⁻¹ nudibranch D⁻¹), mean volume of nudibranch per square meter (mL nudibranch m⁻²), percentage of nudibranchs feeding on H. perlevis, the surface of Lomergat (m²) and the number of days from May to ​ October (i.e.; 184 days). On the other hand, the total volume of H.perlevis in Lomergat ​ ​ ​ ​ was calculated by multiplying the mean biomass of sponge per square meter (mL sponge m⁻²) by the surface of the maerl bed.

• Results

1) Abundance estimation and distribution of H. perlevis and D. verrucosa ​ ​ ​ In June 2020, the mean D.verrucosa abundance was 17.92 ± 11.85 individual ​ ​ m⁻² and the mean biomass was 4.10 ± 3.09 mL m⁻², where 94.70% of D.verrucosa ​ were observed feeding on H. perlevis. As for the sponges, the mean H. perlevis ​ ​ ​ abundance in June 2020 was 23.77 ± 12.78 individual m⁻² and the mean biomass was 269.65 ± 164.28 mL m⁻². On the other hand, in November 2020, the mean D.verrucosa abundance was ​ ​ 0.923 ± 1.29 individual m⁻² and the mean biomass was 0.230 ± 0.432 mL m⁻². As for the sponges, the mean H. perlevis abundance in November 2020 was 20.31 ± 13.47 ​ individual m⁻² and the mean biomass was 20.98 ± 22.12 mL m⁻². ​ Both abundance and biomass of D. verrucosa noticeably decreased from June ​ 2020 (active season) to November 2020 (non-active season). For sponges, there was a net decrease of the biomass from June to November 2020 while the H. perlevis ​ abundance remained similar.

2) Predation activity: characteristic and rates

Intra-individual and inter-individual variabilities

The laboratory experiment has been led on 30 nudibranchs and repeated 5 times. The 5 repetitions permitted to determine the intra-individual variability while the 30 subjects enabled to establish the inter-individual variability.

No specific intra-individual variability was observed along the experiment repetitions. Nudibranchs did not show any specific pattern on sponge selection. No correlation was found between the nudibranch and the amount of sponge eaten (Fig.4). ​ ​ However, nudibranchs were not eating on sponges on 5/5 experiments. As the study does not focus on nudibranch behaviour, we were not able to determine which factors were responsible for the nudibranchs feeding activity. On the other hand, there is inter-individual variability in the quantity of sponge eaten in 24h (mL sponge D⁻¹) and which can be explained by the nudibranchs size. We found a robust correlation between the volume of sponge eaten per day and the volume of the nudibranchs (Fig.5). Only 35 ​ ​ ​ nudibranchs were used to calculate this correlation since 5 nudibranchs, out of the 40 used, did not feed on sponges along the experiments. As the statistical figures were relevant (n=35, R²= 0.7477 , p-value=1.061 × 10⁻¹¹; Fig.5), we can say that the volume ​ ​ of sponge eaten per day depends on the nudibranch volume. In other terms, a large nudibranch will eat a greater amount of sponge than a small nudibranch. This variability did not come from the growth of nudibranchs during the experiments because the 5 experiment repetitions along an experimental set only extended over 2 weeks, which is too short to detect a significant growth in nudibranchs [25]. ​ ​

Predation rate

Given that the volume of sponge eaten per day depends on nudibranch volume, we decided to estimate the predation rate as the volume of sponge eaten per volume of nudibranch and per day (mL sponge mL⁻¹ nudibranch D⁻¹). Therefore, this predation rate is reliable for nudibranchs of all sizes.

After testing the relation between predation rate and the volume of the sponge given during the experiment, we found that the predation rate is slightly influenced by -13 the sponge volume (n=81, R²= 0.4993, p-value=1.198 10 ;​ Fig.6). Therefore, we can ​ ​ ​ say that nudibranchs tend to have a higher predation rate on larger sponges than on smaller ones. In fact, small sponges (<5mL) were often totally eaten at the end of the experiment, consequently the predation rate was limited by the small volume of the sponge itself.

The mean predation rate of the laboratory experiments is 0.326 ± 0.143 mL sponge mL⁻¹ nudibranch D⁻¹. On the other hand, the mean predation rate of the in situ experiment was 0.156 ± 0.091 mL sponge mL⁻¹ nudibranch D⁻¹ (Fig.7). The studied ​ ​ D.verrucosa from both laboratory and in situ experiments received sponges with similar sizes (Mann-Whitney U test: p-value=0.061>0.05). Therefore, the predation rate differences between the laboratory and in situ experiment was likely due to the period of starvation which was imposed on the laboratory subjects but not on the in situ ones.

3) Impact on sponge’s community (extrapolation)

Considering that the predation rate from both experiments were significantly different and could not be used as an overall data set, only the mean predation rate from the in situ experiments (0.156 ± 0.091 mL sponge mL⁻¹ nudibranch D⁻¹) was used ​ ​ to extrapolate the impact of the D.verrucosa on H.perlevis’s community in the maerl bed ​ ​ of the Bay of Brest.

For the extrapolation, the average biomass of the nudibranchs in Lomergat (mL nudibranch m⁻²) was calculated from their length with the following equation: volume=1.80448 × length (Fig.8) and was estimated as 4.099 ± 3.094 mL nudibranch ​ ​ m⁻².

Firstly, to determined the average biomass of sponge eaten per day and per square meter (mL sponge D⁻¹ m⁻²) we multiplied the average biomass of nudibranchs in Lomergat (4.099 ± 3.094 mL nudibranch m⁻²) and the average in situ predation rate (0.156 ± 0.091 mL sponge mL⁻¹ nudibranch D⁻¹). Given that 94.70% of the individual of D.verrucosa were observed eating at the natural habitat, the biomass of H.perlevis ​ eaten per day in Lomergat was estimated to be 0.604 ± 0.577 mL D⁻¹ m⁻².

Secondly, to extrapolate the total biomass of H.perlevis eaten to the Lomergat’s ​ area, we multiplied biomass of H.perlevis eaten per day (0.604 ± 0.577 mL D⁻¹ m⁻²) by ​ the surface of Lomergat (3810000 ± 100 m²). Therefore, the mean volume of sponge eaten per day in Lomergat is 2.30 ± 2.20 m³ D⁻¹.

Thirdly, the volume of sponge eaten in Lomergat from May to October by multiplying the mean volume of sponge eaten per day by the number of days during the active season of D. verrucosa (184 days). Therefore, the average volume of sponge eaten in Lomergat from June to October is 423.65 ± 404.80 m³.

Finally, given the total volume of H.perlevis in Lomergat is 1027.37 ± 625.92 m³, ​ ​ D. verrucosa ate 41.24 ± 25.12% of the overall H. perlevis population from May to ​ October. We can conclude that the D. verrucosa population heavily impacts the H. ​ ​ perlevis population in Lomergat, and subsequently its dynamic. ​

In November 2020, the biomasses estimation H. perlevis was 20.98 ± 22.12 mL ​ m⁻². In comparison with the data from June 2020 (see part “Abundance estimation” in the Results section), we observed a decrease of 92.22% of the H. perlevis biomass in ​ ​ ​ the maerl bed of Brest. Given that D. verrucosa ate 41.24 ± 25.12% , their predation is ​ not the only factor impacting the H. perlevis distribution. ​ ​

• Discussion

H. perlevis is the most abundant sponge species in the Bay of Brest and represents most of the sponge biomass in the maerl beds of the Bay [17]. In the maerl ​ ​ bed of Lomergat, we estimated its average biomass as 269.65 ± 164.28 mL m⁻². In 2018, its biomass was estimated by López-Acosta et al. as 173.9 ± 236.6 mL m⁻² [17] in ​ the different maerl beds of the Bay of Brest. Given that the same quadrat method was used, this study showed a H. perlevis biomass significantly lower than what we ​ estimated. This difference could be explained by the monitoring area. In fact, in her study, López-Acosta led her experiments in the overall maerl beds of the Bay while in this study, the sponge transects were made in the specific maerl bed area of Lomergat which have the highest numbers of sponge abundance and biomass of the Bay, hosting a rich and overflowing sponge community.

Our results indicated that the predation of D. verrucosa has an effect on the ​ biomass and dynamics of the sponge H. perlevis in the Bay of Brest. Among others, this ​ activity has a significant impact on the role of this species to the silicon budget of the Bay. The individuals of H. perlevis have a specific silicon content of 48.88 ± 6.18 mg of ​ biogenic silica (BSi) ( SiO ) per mL of sponge [26]. From May to October 2020, D. 2 ​ ​ ​ ​ verrucosa eliminated 20707.81 ± 108.39 kg of BSi within the population of H. perlevis in ​ ​ the total area of Lomergat, which is equivalent to 5.435 ± 0.028 g BSi m⁻². In term of silicon, we can say that nudibranchs consumed 2.541 ± 0.013 g Si m⁻² from May to October. López-Acosta et al. (2018), estimated that the regular silicon consumption of ​ H. perlevis ―when individuals are not disturbed― in the maerl beds of the Bay of Brest ​ ​ was 6.45 ± 7.65 mmol Si m⁻² in 6 months [17] which is equivalent to 0.181 ± 0.215 g Si ​ m⁻². Hence, the nudibranch predation is almost 3 orders of magnitude superior than the H. perlevis regular silicon consumption. Given that the biomass of H. perlevis seems to be stable from one year to another according to the database of the long-term survey conducted by the Observatoire de Fauna et Flora of IUEM, the rate of recovery of H. ​ ​ perlevis is likely fastened to regenerate the amount of tissues consumed by the ​ nudibranch predation during the active predatory season (i.e., May – October). This phenomenon has also been observed in other sponges, which showed growth rates orders of magnitude higher than undisturbed individuals to regenerate their damaged tissues [24]. This has a major impact on the role of sponges in the silicon cycle of the ​ ​ Bay of Brest (and potentially in other marine ecosystems where predation activity is high). To date, sponges have been considered to slightly contribute to the silicon fluxes due to their slow growth, long living features. However, if H. perlevis recovers its ​ ​ biomass every year during the season that is not predated (November – April), this would mean that sponges would be consuming a significant larger amount of silicon from the seawater of the Bay that was primarily estimated, when neither natural nor anthropogenic disturbances were considered in the sponge community. In addition to that, the predation activity of D. verrucosa accelerates the release of sponge BSi to the sediments. In fact, the spicules ingested by the nudibranch are eliminated with the fecal pellets which facilitate the release of sponge BSi into the sediments before the sponge dies.

In the sponge predation literature, most of the studies agreed on the fact that spongivore predators have an influence on the distribution and dynamics of sponges, either the study was led on tropical latitudes or in high latitudes. We obtained similar results as Dayton [9] and Pawlik [8] in which they respectively led their study in ​ ​ Antarctica and on the Caribbean coral reefs. As we obtained a 41.24 ± 25.12% decrease on the H. perlevis community due to D. verrucosa predation, Dayton obtained a 42.9% ​ ​ decrease of the Rossella racovitzae abundance due the predation of the Antarctic ​ nudibranch species Austrodoris mcmurdensis. Dayton’s study can be relevantly ​ ​ compared to ours knowing that the predation rate was estimated by the same method: measuring the biomass of sponge eaten by the predators. On the other hand, Pawlik obtained a decrease superior to 50% of the coral reef sponges community due to the ​ tropical predator fishes. Although we obtain similar results, Pawlik estimated the sponges biomass eaten by measuring the wet weight before and after predation. As I precised in the Material and methods section, most of the sponges, including H. ​ ​ perelvis, do not support being out of the water. Pawlik mentioned a significant and ​ inexplicable weight loss for certain sponges during the experiment. This experience could have led to an inaccuracy of the predation rate given that sponges risk being affected by the wet weight measurement but there were no specific indications concerning this potential bias. Moreover, in their review from 2020, Pawlik&McMurray [29] made the statement that the only valid indicators for sponge predation are gut content measurement and volume ingested by predation quantification. To date, the predation rate estimation by counting the number of bites taken on the sponge [4,30] or ​ by measuring the wet weight ingested by predators [8] are considered skewed. ​ ​ However, even if sponge predation literature agrees on the influence of predation on the sponge community, the magnitude of the predation’s impact varies regarding the predator. Indeed, Sheild&Witman published a study about the impact of the sea star Henricia sanguinolenta on the finger sponge Isodictya spp in Northern Massachusetts ​ ​ and observed a sponge biomass decrease of 78.6% along 1.5 years [10]. This high ​ ​ impact percentage can mainly be explained by the difference between the predators (sea stars vs nudibranchs), which probably have different feeding requirements. This is supported by another study, in which it was determined that the eating rate of the seastar Oreaster reticulatus varied from 6.3 and 157.08 mL of sponge eaten per day ​ ​ and per individual [5], which is much higher than what we measured. In fact, O. ​ ​ ​ reticulatus are organisms which are much bigger than D. verrucosa. In her study, Wulff ​ ​ ​ worked with O. reticulatus of ± 31cm diameter large. ​ ​

• Conclusion and perspectives

Through this study, we were able to highlight the predator-to-prey relationship between D. verrusoca and H. perlevis and determine the impact of this predation on the ​ ​ ​ sponge population dynamics in the Bay of Brest, by measuring the volume of sponge consumed by the predator. From May to October 2020, D. verrucosa community ate ​ 41.24% ± 0.467 of the sponge H. perlevis community in the maerl bed of Lomergat. ​ However, this study could be deepened by monitoring the biomass of H. perlevis during ​ a year cycle to determine if its growth rate varies depending on the active and non-active predation season of D. verrucosa, and determine how they regenerate their ​ tissue. Moreover, according to Pawlik&McMurray [29], another relevant method to ​ ​ measure predation rates on sponges is by measuring the sponge volume of the ​ predator's gut contents. Leading the same study with different predation quantification methods would permit to compare the results obtained and verify the accuracy of the nudibranch’s impact on the sponges community.

This study could also be deepened by taking into account the impact of the warming disturbances on predation. In fact, the increase of predation rates due to global warming have been widely studied by the research community. For instance, the feeding rates of herbivorous fishes on kelps algaes largely increased in temperate reefs due to climate change and consequently prevented the kelp forest reestablishment [31]. ​ ​ In order to prevent a similar scenario regarding the sponge community, it would be interesting to study the impact of heating waves on spongivore predators, like nudibranchs.

On a wider scale, this project could contribute to a worldwide study such as the comparison of sponge predation depending on latitudes. This could allow to determine where the natural and human related impacts are high and consequently put the sponge community at risk. Given that sponges play lots of essential roles in the well-functioning of the marine ecosystem, this global knowledge could be used to prevent future ecological crises.

• References

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• Supplementary material/ annexes

Annex 2.

The Time Lapse videos can be watched in this link: Time Lapses of the nudibranch ​ activity

Nudibranchs do not find the sponge immediately. Once they find it, they do not always keep feeding on it. As we can see on the laboratory experiment Time Lapses, the nudibranch found the sponge and started feeding on it at the beginning of the experiment (t1h-t2h). On the other hand, it stopped feeding along the experiment and was observed not feeding at the end (t22h-t23h).