Navigating Soundscapes: Attractant effect of reef sound on oyster settlement may be attenuated by vessel noise

Navigating Soundscapes: Attractant effect of reef sound on oyster settlement may be attenuated by vessel noise | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Navigating Soundscapes: Attractant effect of reef sound on oyster settlement may be attenuated by vessel noise Sarah Schmidlin, Clea Parcerisas, Jeroen Hubert, Maryann S. Watson, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3940393/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Settlement is a critical period in the life cycle of benthic species with planktonic larval stages and for reef building invertebrates such as oysters and corals; settlement rates are predictive for reef restoration and long-term survival. Increasing evidence suggests that marine invertebrates use information from ocean soundscapes to inform settlement decisions. Sessile marine invertebrates with a settlement stage are particularly reliant on environmental cues to direct them to ideal habitats as settlement location is permanent. As gregarious settlers, oysters prefer to settle amongst members of the same species. It has been hypothesized that planktonic larvae use distinct oyster reef sounds to navigate to ideal habitats. In controlled laboratory experiments, we show that sounds recorded at conspecific reefs induce higher percentages of settlement in larvae of the Pacific Oyster Magallana gigas . Additionally, we exposed larvae to anthropogenic sounds from several different vessels, combined reef-vessel sounds as well as off-reef and no sound controls. Our results suggest that attractive reef noises may be masked by vessel sounds, however, this observation is substantiated by a nonsignificant trend. Examining the acoustic characteristics of the preferred reef sounds, we hypothesize that spectro-temporal patterns are the driving attractive quality in reef sounds for this species. Biological sciences/Ecology/Behavioural ecology Biological sciences/Ecology/Restoration ecology Physical sciences/Physics/Applied physics/Acoustics Earth and environmental sciences/Ocean sciences/Marine biology Larvae settlement underwater noise noise pollution soundscapes settlement cue oyster reef ecology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Identifying a suitable habitat prior to permanently transitioning to a benthic life stage is critical for future survival, growth and reproduction in many marine invertebrates with planktonic larvae. These species therefore evolved the perception of a variety of environmental cues, enabling them to identify promising settlement locations 1 . Experimental research has shown that in some species, a single cue can induce settlement and subsequent metamorphosis 1,2,3 . But larvae may respond to more than one cue 1 and sometimes even rely on a specific combination of cues 4 . Cues can have chemical and physical origins, and while some types of cues require close contact with a prospective settlement location, other cues may act over larger distances to guide larvae to their preferred habitat 1,5 . As sound propagates relatively fast and far underwater, it serves as an efficient signal transmission medium. For marine species, specific events, such as predator presences or mating opportunities can be understood by the particular sound event associated with that predator or mate 6,7 . However, collectively, soundscapes can also convey the overall quality and suitability of the environment for a species 8,9 . Research on acoustic cues informing larvae about optimal habitats has only been established relatively recently 10,11 . In certain invertebrate species with a settlement/metamorphosis stage, including crabs, corals, and bivalves, acoustic cues have been shown to affect larvae swimming direction 10,11 , settlement propensity 12,11 , and the timing of metamorphosis 13,14,15 . In general, it seems that natural environmental sounds seem to act as a positive cue to the invertebrate species in that environment 16 . In coral and bivalve reefs, larvae seem to be attracted to soundscapes from healthier reefs, which produce louder and more acoustically complex sounds compared to less healthy reefs which are much quieter 12,17 . However, the particular characteristics of the reef soundscape (i.e. sound pressure level (SPL), specific frequencies, complex mixtures of these or other acoustic characteristics) that elicit settlement behaviors remain unclear. Anthropogenic sounds may interfere with or mask natural marine soundscapes 8 . Vessel noise can mask important sound cues and consequently poorer orientation toward reef sounds in fishes 18,19 , and delay settlement in coral larvae (planulae) 20 . Anthropogenic noise can not only disrupt or reduce larval settlement but may also be (mis)interpreted as a positive cue in some taxa 14,15,21,22,23 . Vessel noises have been shown to increase some larvae settlement, including in the mussels Perna canaliculus 14 and Mytilus edulis 22 . Why anthropogenic noises are interpreted as a positive cue in some taxa but are repulsive to others is unknown. The reaction to anthropogenic sounds may depend on the acoustic profile of a species' preferred habitat and which features of this profile are responsible for attraction 15,21,24,25 . The oviparous true oyster Magallana gigas is an important reef-building ecosystem engineer 26 and a valuable species for aquaculture 27 . But in many areas across the globe it is invasive and considered a biofouling pest 28 that poses a threat to local species and ecosystems 29 . There is considerable interest in settlement preferences of this species for both bolstering as well as reducing recruitment 30 . The availability of settlement cues is crucial for reef sustainment, with some reports suggesting that these cues may outweigh other recruitment factors such as local hydrodynamics, and larvae supply 31,32 . The recent revelation that oysters not only settle more rapidly but also exhibit horizontal swimming movements toward sound sources underscores the significance of soundscapes as a navigation tool for larvae 11 . So far, the larvae of M. gigas have not been studied for their response to acoustic settlement cues (but see Stocks et al, (2012) 21 for an account on swimming activity in response to natural and vessel sounds). Other true oysters with relevant experimental data are the closely related and also oviparous Crassostrea virginica, and the more distantly related larviparous Ostrea angasi . Experimental studies have shown that both C. virginica and O. angasi larvae prefer louder reef sounds over more quiet off-reef playbacks or no-sound controls 11,33,34 . In this study, we present the results of laboratory-playback based settlement experiments on the role of acoustic cues in settlement and metamorphosis of Magallana gigas . Firstly, we were interested in the importance of oyster reef sound compared to off-reef sound. Secondly, we wanted to know whether vessel noise attracts or repels pediveliger larvae. To do so, we exposed the larvae to different vessel and reef sounds as well as off-reef and no-sound controls. Finally, we submitted the larvae to vessel and reef sounds simultaneously in order to find out whether vessel noise modifies, or even completely masks oyster reef sound cues. Methods Soundscape measurements All the recordings used during the experiment were recorded in two regions of the North Sea: the Southern Bight near the Belgian coast and in the Dutch Wadden Sea (see Figure 1). Details of the data collection are explained in the Supporting Information (Appendix S1). The recorded data were manually scrolled through to select suitable files for the off-reef and the vessel treatments. Only data from spring and summer were considered, in order to correspond to the sounds from the Wadden Sea (reef and off-reef). Selected data are listed in the Supporting Information Appendix S1, Table S2. In total, 3 recordings of reefs from 2 different locations, 4 vessel recordings with several boats on each recording from 4 different locations, and 4 off-reef recordings from 3 different locations were used to represent our treatments. Sound files The collected sounds were scanned to select appropriate sound snippets. These segments were selected to be representative of each treatment. For example, reef sounds were only selected when they contained no apparent outside influences (e.g. vessel sounds). For the vessel sounds, a fair variability of sounds was selected, from short sounds of distant vessels to longer continuous sounds from vessels operating close by, with no other audible background sounds. The selected segments were then combined to create one 1 h file per treatment and day. In some cases, the selection led to files shorter than 1 h, so the segments were repeated and combined by applying crossfading with Audacity 35 to create a 1 h file. When enough recordings were available for 1 h or more, segments were not repeated. Throughout the experiment, the treatment groups were consistent but the sound file differed in each replicate. For reef treatment, sounds used were recorded from the same location in Texel, NL but sound files used during each day of the experiment were selected from different recording dates (see Table S2). For the off-reef sounds two sound files were used recorded from Texel, NL and two sound files were used recorded from non-reef areas in the Southern Bight off the coast of Belgium (see Table S2). All vessel sounds were recorded from locations in the Southern Bight (see Table S2). Treatments where vessel sounds and reef sounds were played together were created artificially overlaying the reef sound file and the vessel sound file. The use of multiple sound files of the same treatment was used to strengthen confidence that the sounds were representative for the overall soundscape and not for a single event. The files used were acquired with different instruments and at different locations. To deal with the difference in sampling rate and minimum recording frequency, all the files were filtered using a butterworth bandpass filter (N=4) between 20 Hz and 12 kHz. After the filtering, all the files were downsampled or upsampled to 48 ksps to match the playback requirements. Information about the instruments used for recording can be found in Table S1. Broodstock and Larvae Culture Ten mature adult oysters (five females and five males) were purchased from the Guernsey Sea Farms Ltd (Guernsey, UK) and used to produce larvae. Eggs were fertilized by gonad stripping following FAO guidelines 36 . Fertilized eggs were kept undisturbed in flat bottom tanks for 48 hours at 22 °C at a density of ten eggs per ml of filtered seawater (FSW). All seawater used in this experiment was filtered at 0.1 µm and passed through UV light. After 48 hours larvae were sieved over 70 µl nylon mesh, rinsed, and transferred to rearing tanks with FSW. Tanks were aerated and kept at 22 °C for the entire duration of larvae rearing. Every two days larvae were sieved over mesh corresponding to the average size of the larvae and the water in the tanks was changed. Larvae were fed a mixture of fresh microalgae mixture consisting of Chaetoceros muelleri, and Isochrysis galbana (clone T-ISO). For the first 4 days larvae were fed at 40,000 cells/ml water using only I. galbana (clone T-ISO). Days 5-12 larvae were fed C. muelleri, and I. galbana (clone T-ISO) at 100,000 cells/ml at a volume ratio of 1:1. Days 13+ larvae were fed C. muelleri, and I. galbana (clone T-ISO) at 100,000 cells/ml at a volume ratio of 3:1. Larvae entered their pediveliger stage and became competent to settle at 29 days. Larvae were determined for competence when they had a prominently displayed eyespot and larval foot and were sized at 320-350 μm in diameter. Settlement Experiment Design The experiment consisted of five sound treatments: oyster reef sounds, vessel noise, reef sounds with added vessel noise, off-reef sounds, and a no-sound control. Larvae were exposed to each of these treatments in parallel, with trials that lasted 24 hours, these trials were replicated four times over four days. On each day, larvae were assessed for settlement and then discarded. At the start of each experiment day, new pediveliger stage larvae were used. Sound treatments took place in separate tanks. In each tank, 5 jars each containing 10 larvae, were used as subreplicates (see Figure 2). Tank set-up Five 100L tanks were used (49x65x50.5 cm), separated 20 cm from each other on a rack. Each tank sat upon a 4 cm layer of polystyrene to isolate it from the rack and an additional layer of acoustically absorbent foam (25mm thick) between the polystyrene and the tank bottom. The acoustic foam was also placed at the tank sides. Four Lubell UW30 Underwater Speakers with custom-made amplifiers, battery-powered in order to avoid 50 Hz noise, were used. Each speaker was connected to one TASCAM playback device which played on repeat a 1 to 2 h file. No speaker was placed in the no-treatment control (see Figure 2). The speakers were hung in the middle of the tank with ropes so they would not touch the tank walls. Larvae were placed inside 100 ml polystyrene jars and these containers were fixed in the same position in the tank for every day of the experiment. Settlement Assays Oyster larvae were reared in a laboratory scale hatchery in the same facilities as where the experiments were conducted. A detailed account on the larviculture can be found in the Supporting Information. On each day, 10 larvae were gently pipetted randomly into each of the five 100 ml containers per tank and filled with filtered seawater (FSW) and 0.2 grams of oyster shells which could act as a settlement substrate. To get a consistent shell topography, shells were crushed using a hammer and crushed shells were sieved between 1.0 mm and 0.5 mm metal sieve. For each treatment tank, 5 individual containers were used. As all treatments were repeated over 4 consecutive days, 20 jars were used per treatment in total. All trials were conducted in a dark environment at 20 (±1) °C in a climate-controlled room. To avoid any air in cups containing larvae, larvae were placed in the cups and the lid was fixed while the cup was fully submerged in FSW. This step was necessary to prevent any distortion of the sounds due to reflection from air bubbles. All FSW used in the experiment had added microalgae Chaetoceros muelleri , and Isochrysis galbana (clone T-ISO) at 100,000 cells/ml at a volume ratio of 3:1. In a previous study, M. gigas larvae increased swimming when exposed to reef sounds, but only if larvae were fed 21 , thus microalgae were added to our larvae containers. Microalgae were added at the same concentration as used in larvae rearing tanks and food levels were not limiting for the duration of the experiment. On top of each tank, cups were attached to a wooden pole sitting horizontally across the tank. Each larvae jar was attached so that it was in a fixed position for the duration of the experiment, the position of the jar was noted so that the effect from placement in the tank could be ruled out. The wooden pole was isolated from the tank walls with polystyrene to avoid vibration propagation. One of the cups was located directly above the speaker and the other 4 cups were at the same distance from the center of the speaker (see Figure 2). After 24 hours of exposure, larvae metamorphosis was checked using a dissecting microscope and the number of larvae that had cemented themselves to the substrates were counted. Metamorphosis was confirmed by gently blowing water over the larvae with a pipette to ensure that larvae were fixed to the substrate. Playback and sound characteristics For each treatment, a playback volume was chosen so the exposure power spectral density (PSD) would match the sound levels specified in literature as typical of reefs (at 1 m from the seafloor) and off-reefs (at 2 km from the reef) 11,37 . Details of the process done to achieve this are specified in Supporting Information. To quantify the exposure sound level and the acoustic characteristics of each playback, each treatment was recorded using the chosen playback volume for 1 h (experiment files) at 48 ksps. When recording these 1 h files, all four different sound treatments of that batch were on to record possible acoustic crosstalk from the other treatments. These 1 h recordings were used to compute the exposure acoustic metrics for each treatment. The no-sound treatment was also recorded while all the other treatments were on. Furthermore, the room noise was also recorded using the same protocol when no speaker was active. For each treatment, several acoustic features were computed for both the 1 h experiment files and the 1 h field files. Acoustic Complexity Index (ACI), Acoustic Evenness Index (AEI) and Acoustic Diversity Index (ADI) were computed using the maad python package 38 , and the Power Spectrum Density (PSD) was computed using the scipy python package 39 . The average PSD was computed for three different bands by averaging the spectrum density of all the frequency bins included in the specified frequency band. Parameters used to compute each of the features are summarized in Table S3. Both ACI and ADI are proxies to quantify acoustic complexity (the higher the number, the more complex), while low values of AEI represent an even sound and higher values represent more uneven sounds. This is not correlated with the ecological concept of evenness, as acoustic evenness refers to an even distribution of sound energy in different frequency bands, and this can be achieved due to a high biodiversity vocalizing at the same time covering all the frequency bands or by constant broadband sounds such as some anthropogenic sounds 40,41 . Statistical analyses A generalized linear mixed-effect model was created using the glmer function of the lmer package 42 in R version 4.1.3 (2022-03-10) (R Core Team, 2021). As the binary response variable was binary (settled vs. not settled) we fitted a Bernoulli distribution using a logit link function. The assumptions of the model were met. N value was between 175 and 180 for each treatment. The sound treatment was the only main effect variable. Individual speaker-playback, the tank used, the position of the ‘larvae container’ within the tank, and the date of the trial (as a factor) were added as main effects to the generalized linear models to determine whether these confounders had any effect on settlement. As there was no significant main effect of speaker, tank, or cup position, it was assumed that they did not have an effect on the experiment outcome and were not included in the final model. The effect of date of the experiment was significant and therefore included as a random effect variable in the final model. Post hoc tests were performed using the emmeans function of the lsmeans package 43 to calculate the marginal means adjusting p-values for multiple comparisons with Tukey's method and the pairs function was used to display pairwise comparisons. Post hoc analyses were also conducted on models where experiment day was included as a main effect variable in order to ensure that this variable did not interfere with the treatment. Results Settlement rate Larvae settled increased 1.40 times in response to reef sound compared to vessel sounds (β = 0.720, SE = 0.217, p = 0.011), 1.41 times compared to off-reef sounds (β = 0.710, SE = 0.218, p = 0.010), and 1.65 times compared to the no sound treatment (β = 0.971, SE = 0.219, p = 0.0001; Table 1 ). When vessel sound was added to the reef sound, the settlement propensity decreased 1.28 times compared to the pure reef sound (β = 0.540, SE = 0.215, p = 0.088), and was 1.10 times higher than in the vessel-only sound treatment (β = 0.162, SE = 0.218, p = 0.946). Comparisons among other treatments revealed only minor differences (Table 1 ). Vessels and off-reef sounds had very similar effects on settlement. The lowest settlement rates were observed in a no-sound control treatment. Model predictions are plotted in Fig. 3 . Table 1 The results of the posthoc of the GLMER model 1 using all data and comparing all treatments. Significant values (p ≤ 0.05) are in bold , while trends (p ≤ 0.10 & > 0.05) are in italic . Contrasting treatments Estimate SE df z.ratio p.value reef - off reef 0.70952 0.218 Inf 3.260 0.0098 reef - vessel 0.70229 0.217 Inf 3.238 0.0106 reef - no sound 0.97083 0.219 Inf 4.440 0.0001 reef - (reef + vessel) 0.54007 0.215 Inf -2.510 0.0884 off reef - vessel 0.00723 0.220 Inf 0.033 1 off reef - no sound 0.26131 0.221 Inf -1.180 0.7629 off reef - (reef + vessel) -0.16945 0.218 Inf 0.776 0.9375 no sound - vessel -0.26855 0.221 Inf 1.216 0.7418 no sound - (reef + vessel) -0.43076 0.219 Inf 1.966 0.2825 vessel - (reef + vessel) -0.16222 0.218 Inf -0.746 0.9457 Playback The recorded sound in the tanks did not perfectly match the spectrum of the sounds recorded in the field due to the technical limitations of the reproduction equipment and the resonances that inevitably occur in tank-based experiments (Fig. 4 ). However, the same patterns were observed when computing for different acoustic metrics (Fig. 5 ). Discussion The results of our laboratory experiment showed a clear attracting effect of sounds of conspecific reefs for larvae of the oyster Magallana gigas . Settlement increased about 1.41 and 1.65 times under the oyster reef treatment compared to off-reef and no sound control treatments, respectively. We further show, for the first time in any oyster species, that vessel sound might mask oyster reef sound, effectively reducing the latter’s attractive effect. Preference for oyster reef sounds Larvae of M. gigas consistently settle more readily when exposed to sounds of reefs inhabited by conspecifics. Our finding thus corroborates earlier research in fish, corals, and other oyster species, where larvae were found to increase settlement or orient more readily towards playback of reef sounds 10 , 33 , 44 , 45 . Yet the sound features that trigger this response still remain to be identified. In general, oyster and coral reefs exhibit higher levels of sound and greater acoustic diversity than off-reef counterparts, due to increased soniferous biological activity including vocalizations of soniferous fishes and invertebrates, both passive or active, as well as the physical complexity of the reef 12 , 20 . It remains undecided in the literature if larvae are able to distinguish particular sounds from different habitats, or if there is simply a preference for certain acoustic features such as SPL 17 . The spectrum of reef sounds recorded for our study followed patterns similar to other oyster reefs 11 , 12 , and were half the time louder, had consistently a higher acoustic complexity, and higher evenness (lower AEI value) than off-reef areas. Compared to the vessel sounds, however, our reef sounds tended to have similar or lower PSD (depending on the vessel). Reef sounds were unique amongst the other treatments in their diversity, with consistently higher ACI and ADI values, and lower AEI values. This indicates that loudness (SPL) alone is not responsible for larval attraction, instead spectro-temporal patterns responsible for a high ACI may play a more important role. This conclusion can be corroborated by reviewing trends in the literature related to the effect of anthropogenic noise on larvae. Pine et al., (2016) 15 found similarly that spectral composition rather than SPL is more relevant in the attraction of crab megalopae to native habitat sounds, as crab megalopae reduce metamorphosis (in comparison to natural habitat sounds) when exposed to wind turbine noise but when the same turbine noises were played back at higher SPL, this did not result in any further changes to crab metamorphosis time. For scallop larvae, Gigot et al., (2023) 23 found different responses from two sources of anthropogenic noise, larvae reduced metamorphosis during drilling sounds but increased when exposed to pile driving sounds. As both sounds were substantially louder than the no sound control, this further indicates the importance of temporal and spectral composition over preference for loud sounds. Preference for louder sounds should not be ruled out completely, Wilkens et al. (2012) 14 found that when exposed to (the same) vessel sound at increasingly louder SPLs, mussel larvae increased settling at the louder treatments. Lillis et al., (2016) 17 also conclude that louder reefs attract more coral settlers than quieter reefs. It is reasonable to conclude that both of these sound qualities (loudness and spectro-temporal patterns) are perceptible to larvae, the preferences for each seem to be highly species-specific and could be based on the preferred habit qualities. Sound masking by vessel noise We found that the addition of vessel noise to the oyster reef sound led to reduced settlement in M. gigas . This finding could be interpreted in one of two ways. Vessel sounds could be masking the attraction-effects of the reef sounds. Alternatively, vessel sounds could have some intrinsically repulsive component for larvae. Given that vessel sounds alone do not reduce settlement compared to sounds from off-reef areas, acoustic masking of preferred habitat sounds seems to be the more parsimonious explanation. Holles et al. (2013) 18 similarly found that fish larvae will avoid swimming toward reef and vessel sounds together more often compared to reef sounds, but did not consistently avoid vessel sounds alone more than the no sound control. They concluded that the larvae might have already experienced boat noise in the ocean and become conditioned to it, or simply because boat noise in the ocean does not represent a need to change their behavioral responses. However, in coral planulae vessel noises do not only mask habitat sounds but also independently produce a negative reaction. Vessel sounds decrease the attraction of coral larvae planulae to chemical cues associated with their preferred habitats 20 . In comparison to other oyster species, our results corroborate well with the results from McAfee et al. (2022) 34 , who found that enrichment of experimental oyster reefs with reef sound in the field was ineffective in an area with high anthropogenic background noise. Future works should confirm the responses of oysters seen here, under vessel noises, including adding vessels of different origins and ranges of spectrum level (SPL). Regardless if larvae are disturbed by anthropogenic sounds by masking or direct avoidance of the sound, these disruptions to settlement, force larvae to increase their pelagic period, resulting in increased predation risk or transport to non-ideal locations. The masking effect from vessels is only established in our study with a non-significant trend (p = 0.08). The effect size, however, is substantial with larvae being 1.28 times less likely to settle when vessel noise is added to the oyster reef sound, which is similar to the vessel noise - oyster reef comparison (reduction by 1.40 times). Treatments of reef and vessel noises together also did not differentiate significantly from off-reef and no sound controls. It is clear that the addition of vessel noises changed the characteristics of the reef sounds in our recorded files. We observed a drastic decrease of ACI, a mild decrease of ADI, and an increase of AEI, when vessel noises were added to reefs. Therefore, even though larval reaction remains uncertain, this shows that vessel noise masks the complexity of reef sounds, possibly in a way that larvae cannot distinguish them anymore. Sound detection by larvae Aquatic invertebrates, including oysters, sense particle motion rather than sound pressure 16 . However, particle motion currently remains challenging to quantify, especially in small tanks. In the free field, sound pressure and particle motion are related, but that is not the case when close to reflecting and pressure relieving surfaces. Therefore, in smaller spaces such a tank, the sound propagation will not necessarily be related to particle motion because the walls and surface will act as pressure release surfaces 46 . Hence, sound pressure measurements can be a poor indicator of the particle motion levels, especially close to the tank walls. However, the magnitude and direction of the particle motion are expected to differ substantially from the one the larvae would experience in the field. This does not pose a problem for the current experiment, as our target was a proof of concept study into whether the settlement rate presented any differences when exposed to different acoustic stimuli in lab conditions, and to do so a fully controlled environment is necessary. Furthermore, the fluctuations over time at a large enough time scale represented in the ACI values are probably not affected by the dynamics of the tank resonances. Hence this clue remains valid. Nonetheless, future research would be necessary to confirm our results under field conditions where particle motion and sound pressure have a more direct relationship. However, in the field, it is currently possible to control added sound, but not possible to remove other background sounds. Implications for reef restoration Due to widespread habitat destruction, oyster reefs have been diminishing globally 30 . As oysters are gregarious organisms, relying heavily on the presence of conspecifics to initiate settlement, reef destruction leads to the loss of individuals, further decreasing the chance of successful recruitment and inhibiting reef regrowth. Classic oyster reef mitigation and restoration projects focus on providing new hard substrates for wild larvae to settle, as well as supplying new adults to reefs, however, the importance of acoustic cues may be overlooked. Recently McAfee et al., (2022) 34 used underwater speakers to enhance soundscapes on constructed reefs resulting in greater initial settlement of the oyster Ostrea angasi . If similar acoustic preferences are established for other species, these same techniques could be employed elsewhere. Our results indicate that M. gigas also responds to acoustic cues, and thus may respond positively to acoustic enrichment as a restoration strategy. Our study also indicates that disruptions to natural habitat sounds from anthropogenic sources may hinder oyster settlement. The acoustic profile of an area should therefore be considered when choosing restoration sites. Conclusion We show that M. gigas larvae will settle more readily in response to the sounds of oyster reefs. The reef sounds were unique in being very acoustically diverse (high ACI), while other acoustic features, such as SPL varied among treatments. This indicates that oyster larvae may be able to detect complex spectro-temporal patterns in the soundscape rather than rely solely on SPL. Furthermore, we find that noises from vessels do not repel larvae any more than the effect of off-reefs sounds or no sound controls. Rather, vessel noises seem to mask attractant reef sounds. While acoustic enrichment may bolster recruitment in oyster reefs, our results suggest that it may be less effective in areas with high levels of vessel noise pollution. We call for more research to replicate our findings in the laboratory and also to incorporate tests for sound masking by vessel noise in field experiments. More quantitative evidence is needed to estimate the critical threshold at which vessel noise may negatively affect oyster recruitment in ecologically realistic settings in the field. Declarations Author contributions SS, CP, JH, and PH conceived the ideas and designed the methodology; MSW, ED, and CP collected the sound data; SS and CP collected the experiment data; SS and PH analyzed the data statistically; CP analyzed the acoustic data; SS and CP led the writing of the manuscript with contributions from all other co-authors. Acknowledgements Part of this project was funded by the Research Foundation Flanders (FWO) as part of the Belgian contribution to the LifeWatch ESFRI. We would like to thank the crew of the RV Simon Stevin for the help with acquiring the acoustic data. This study would have not been possible without the lending of the playback equipment from Leiden University. The authors would also like to thank Mattias Bossaer for his help with the lab set-up. Funding for the reef recordings in the Netherlands part of the Swimway Waddenzee project; financial contributions from the Waddenfund, the Ministry of Agriculture, Nature Management and Food Safety, and the Dutch provinces Noord-Holland, Groningen and Friesland. Conflicts of interest The authors state that there are no conflicts of interest pertaining to the research, authorship, and publication of this article. There are no financial, personal, or professional associations that might have influenced or could be perceived to have influenced the research, findings, or interpretations presented in this manuscript. The authors affirm that the content of this paper is based solely on the objective analysis and presentation of the research data. Data availability statement All data supporting the results in this paper are located in a Github repository. https://github.com/sschmidlin/larvae-and-sound References Hadfield, M., & Paul, V. Natural Chemical Cues for Settlement and Metamorphosis of Marine-Invertebrate Larvae. Marine Chemical Ecology, 431–461 (2001). Burke, R. D. Pheromonal Control of Metamorphosis in the Pacific Sand Dollar, Dendraster excentricus. Science 225, 442–443 (1984). Pearce, C. M. & Scheibling, R. E. Induction of Metamorphosis of Larvae of the Green Sea Urchin, Strongylocentrotus droebachiensis , by Coralline Red Algae. The Biological Bulletin 179, 304–311 (1990). Chia, F.-S. & Koss, R. Induction of Settlement and Metamorphosis of the Veliger Larvae of the Nudibranch, Onchidoris bilamellata. International Journal of Invertebrate Reproduction and Development 14, 53–69 (1988). McAfee, D. et al. Soundscape enrichment enhances recruitment and habitat building on new oyster reef restorations. Journal of Applied Ecology 60, 111–120 (2022). Amorim, M. C. P., Vasconcelos, R. O. & Fonseca, P. J. Fish Sounds and Mate Choice. in Sound Communication in Fishes (ed. Ladich, F.) 1–33 (Springer, Vienna, 2015). Hughes, A. R., Mann, D. A. & Kimbro, D. L. Predatory fish sounds can alter crab foraging behaviour and influence bivalve abundance. Proceedings of the Royal Society B: Biological Sciences 281, 20140715 (2014). Duarte, C. M. et al. The soundscape of the Anthropocene ocean. Science 371, eaba4658 (2021). Miller, P. J. O. et al. Behavioral responses to predatory sounds predict sensitivity of cetaceans to anthropogenic noise within a soundscape of fear. Proceedings of the National Academy of Sciences 119, e2114932119 (2022). Vermeij, M. J. A., Marhaver, K. L., Huijbers, C. M., Nagelkerken, I. & Simpson, S. D. Coral Larvae Move toward Reef Sounds. PLOS ONE 5, e10660 (2010). Williams, B. R., McAfee, D., & Connell, S. D. Oyster larvae swim along gradients of sound. Journal of Applied Ecology, 59 (7), 1815–1824 (2022). Lillis, A., Eggleston, D. & Bohnenstiehl, D. Estuarine soundscapes: distinct acoustic characteristics of oyster reefs compared to soft-bottom habitats. Mar. Ecol. Prog. Ser. 505, 1–17 (2014). Stanley, J. A., Radford, C. A. & Jeffs, A. G. Induction of settlement in crab megalopae by ambient underwater reef sound. Behav. Ecol. 21, 113–120 (2010). Wilkens, S. L., Stanley, J. A. & Jeffs, A. G. Induction of settlement in mussel (Perna canaliculus) larvae by vessel noise. Biofouling 28, 65–72 (2012). Pine, M. K., Jeffs, A. G., Wang, D. & Radford, C. A. The potential for vessel noise to mask biologically important sounds within ecologically significant embayments. Ocean & Coastal Management 127, 63–73 (2016). Solé, M. et al. Marine invertebrates and noise. Frontiers in Marine Science 10, (2023). Lillis, A., Bohnenstiehl, D., Peters, J. W. & Eggleston, D. Variation in habitat soundscape characteristics influences settlement of a reef-building coral. PeerJ 4, e2557 (2016). Holles, S., Simpson, S., Radford, A., Berten, L. & Lecchini, D. Boat noise disrupts orientation behaviour in a coral reef fish. Mar. Ecol. Prog. Ser. 485, 295–300 (2013). Wilson, L., Constantine, R., Pine, M. K., Farcas, A. & Radford, C. A. Impact of small boat sound on the listening space of Pempheris adspersa, Forsterygion lapillum, Alpheus richardsoni and Ovalipes catharus. Sci Rep 13, 7007 (2023). Lecchini, D. et al. Boat noise prevents soundscape-based habitat selection by coral planulae. Sci Rep 8, 9283 (2018). Stocks, J. R. Response of Marine Invertebrate Larvae to Natural and Anthropogenic Sound: A Pilot Study. TOMBJ 6, 57–61 (2012). Jolivet, A. et al. Validation of trophic and anthropic underwater noise as settlement trigger in blue mussels. Sci Rep 6, 33829 (2016). Gigot, M. et al. Pile driving and drilling underwater sounds impact the metamorphosis dynamics of Pecten maximus (L., 1758) larvae. Marine Pollution Bulletin 191, 114969 (2023). Erbe, C. Critical ratios of beluga whales ( Delphinapterus leucas ) and masked signal duration. The Journal of the Acoustical Society of America 124, 2216–2223 (2008). Li, S. et al. Mid- to high-frequency noise from high-speed boats and its potential impacts on humpback dolphins. The Journal of the Acoustical Society of America 138, 942–952 (2015). Walles, B. et al. From artificial structures to self-sustaining oyster reefs. Journal of Sea Research 108, 1–9 (2016). Botta, R., Asche, F., Borsum, J. S. & Camp, E. V. A review of global oyster aquaculture production and consumption. Marine Policy 117, 103952 (2020). McAfee, D. & Connell, S. D. The global fall and rise of oyster reefs. Frontiers in Ecology and the Environment 19, 118–125 (2021). Ruesink, J. L. et al. Introduction of Non-Native Oysters: Ecosystem Effects and Restoration Implications. Annual Review of Ecology, Evolution, and Systematics 36, 643–689 (2005). Beck, M. W. et al. Oyster Reefs at Risk and Recommendations for Conservation, Restoration, and Management. BioScience 61, 107–116 (2011). Pineda, J., Porri, F., Starczak, V. & Blythe, J. Causes of decoupling between larval supply and settlement and consequences for understanding recruitment and population connectivity. Journal of Experimental Marine Biology and Ecology 392, 9–21 (2010). Koehl, M. A. R. & Cooper, T. Swimming in an Unsteady World. Integrative and Comparative Biology 55, 683–697 (2015). Lillis, A., Eggleston, D. B. & Bohnenstiehl, D. R. Oyster Larvae Settle in Response to Habitat-Associated Underwater Sounds. PLOS ONE 8, e79337 (2013). McAfee, D. et al. Soundscape enrichment enhances recruitment and habitat building on new oyster reef restorations. Journal of Applied Ecology 60, 111–120 (2022). Team A. Audacity(R): Free Audio Editor and Recorder [Computer application];1999–2019. (2023). Helm, M. M., Bourne, N., Lovatelli, A. (comp /ed), & Fisheries and Aquaculture Management Division. The Hatchery Culture of Bivalves: A Practical Manual . (FAO, Rome, Italy, 2004). Lillis, A., Bohnenstiehl, D. R. & Eggleston, D. B. Soundscape manipulation enhances larval recruitment of a reef-building mollusk. PeerJ 3, e999 (2015). Ulloa, J. S., Haupert, S., Latorre, J. F., Aubin, T. & Sueur, J. scikit-maad: An open-source and modular toolbox for quantitative soundscape analysis in Python. Methods in Ecology and Evolution 12, 2334–2340 (2021). Virtanen, P. et al. SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat Methods 17, 261–272 (2020). Bradfer-Lawrence, T. et al. Guidelines for the use of acoustic indices in environmental research. Methods in Ecology and Evolution 10, 1796–1807 (2019). Mammides, C., Goodale, E., Dayananda, S. K., Luo, K., & Chen, J. On the use of the acoustic evenness index to monitor biodiversity: A comment on “Rapid assessment of avian species richness and abundance using acoustic indices” by Bradfer-Lawrence et al. (2020) [Ecological Indicators, 115, 106400]. Ecological Indicators , 126 , 107626. (2021) Bates, D., Mächler, M., Bolker, B. & Walker, S. Fitting Linear Mixed-Effects Models Using lme4. Journal of Statistical Software 67, 1–48 (2015). Lenth, R. V. Least-Squares Means: The R Package lsmeans. Journal of Statistical Software 69, 1–33 (2016). Montgomery, J. C., Jeffs, A., Simpson, S. D., Meekan, M. & Tindle, C. Sound as an Orientation Cue for the Pelagic Larvae of Reef Fishes and Decapod Crustaceans. in Advances in Marine Biology vol. 51 143–196 (Academic Press, 2006). Radford, C. A., Stanley, J. A., Simpson, S. D. & Jeffs, A. G. Juvenile coral reef fish use sound to locate habitats. Coral Reefs 30, 295–305 (2011). Rogers, Peter H., et al. "Parvulescu revisited: small tank acoustics for bioacousticians." The effects of noise on aquatic life II . Springer New York, (2016). Additional Declarations No competing interests reported. Supplementary Files NavigatingSoundscapesSupplementaryInformation.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 04 Apr, 2024 Reviews received at journal 29 Mar, 2024 Reviewers agreed at journal 20 Mar, 2024 Reviewers agreed at journal 01 Mar, 2024 Reviewers invited by journal 01 Mar, 2024 Editor assigned by journal 28 Feb, 2024 Editor invited by journal 25 Feb, 2024 Submission checks completed at journal 25 Feb, 2024 First submitted to journal 08 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3940393","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":274786665,"identity":"b6aa74e1-3963-4e3c-95ff-7f97be4aab2c","order_by":0,"name":"Sarah Schmidlin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIie2QsUrDUBSG/8OFdAm4lUrBvMIJHSooPssNQjIVOjqFWwLp2FXxJTLWLXDWuAfiYBEyRxwVNBXpINzY0eF+w+Ge4eP8/wUcjn+K2g8Gmckz4wwjMv37WEUzZlDHK8BEA5HZr0NKsH7cvS2RBvOxrGq9fEoKUVlH20urwlUym95Cwof7KDvX3C4KIcNUxXYFMaY+Ss1NlPddZHGXkdGUiz3YplXvPtKDkpz2Skn5p71MHXv9FXVQ9ImilaG8tAerW+/CZwmL5ruLhBtFGaLqeiBYrBr/Jg24SXZ19yGBN1q/dK/bK3uwn0/4hf5DcDgcDscwX/M8Us7YDlIyAAAAAElFTkSuQmCC","orcid":"","institution":"Flanders Marine Institute (VLIZ)","correspondingAuthor":true,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Schmidlin","suffix":""},{"id":274786666,"identity":"65c5761f-b8a9-454b-b3ae-d38b5564ea4e","order_by":1,"name":"Clea Parcerisas","email":"","orcid":"","institution":"Flanders Marine Institute (VLIZ)","correspondingAuthor":false,"prefix":"","firstName":"Clea","middleName":"","lastName":"Parcerisas","suffix":""},{"id":274786667,"identity":"30a78a7b-7905-43a0-962d-2155a712f6a6","order_by":2,"name":"Jeroen Hubert","email":"","orcid":"","institution":"Leiden University","correspondingAuthor":false,"prefix":"","firstName":"Jeroen","middleName":"","lastName":"Hubert","suffix":""},{"id":274786670,"identity":"f1b67383-e88b-446d-828c-a752ac144527","order_by":3,"name":"Maryann S. Watson","email":"","orcid":"","institution":"University of Groningen","correspondingAuthor":false,"prefix":"","firstName":"Maryann","middleName":"S.","lastName":"Watson","suffix":""},{"id":274786672,"identity":"08429216-e0fc-4039-a209-bb4ca51d3aed","order_by":4,"name":"Jan Mees","email":"","orcid":"","institution":"Flanders Marine Institute (VLIZ)","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Mees","suffix":""},{"id":274786673,"identity":"2377154b-8eac-4d06-b77e-6fe588f227b9","order_by":5,"name":"Dick Botteldooren","email":"","orcid":"","institution":"Ghent University","correspondingAuthor":false,"prefix":"","firstName":"Dick","middleName":"","lastName":"Botteldooren","suffix":""},{"id":274786674,"identity":"a9708b80-6b12-4cef-87af-2554ba104d46","order_by":6,"name":"Paul Devos","email":"","orcid":"","institution":"Ghent University","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Devos","suffix":""},{"id":274786676,"identity":"35b4a5a9-a422-4d42-8de7-a23cd4a9e166","order_by":7,"name":"Elisabeth Debusschere","email":"","orcid":"","institution":"Flanders Marine Institute (VLIZ)","correspondingAuthor":false,"prefix":"","firstName":"Elisabeth","middleName":"","lastName":"Debusschere","suffix":""},{"id":274786677,"identity":"63e71eed-11bd-4047-b930-6608579d519a","order_by":8,"name":"Pascal I. Hablützel","email":"","orcid":"","institution":"Flanders Marine Institute (VLIZ)","correspondingAuthor":false,"prefix":"","firstName":"Pascal","middleName":"I.","lastName":"Hablützel","suffix":""}],"badges":[],"createdAt":"2024-02-08 15:30:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3940393/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3940393/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51680922,"identity":"7c34d543-061e-46fd-933a-90a8cc1e245e","added_by":"auto","created_at":"2024-02-27 06:37:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":92069,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of the locations where underwater sound data were collected. Colors represent which treatment was collected there. The round shape represents data collected in the Southern Bight and the square shape represents data collected in the Wadden Sea. Sounds acquired in locations with two colors were used for different treatments. Map made by maps@vliz\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3940393/v1/8bfbe6c46ccb73152f770fb0.png"},{"id":51680923,"identity":"0468289e-1c5f-4d02-bc60-c86faf77c2f9","added_by":"auto","created_at":"2024-02-27 06:37:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":94190,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic depicting five tanks. Four of these tanks are equipped with speakers, each of which is connected to a playback device. All speakers and playback devices are linked to a DC battery as their power source. Five 100ml jars were securely positioned to hang at the same height above the speakers. The speakers were suspended within the tanks in a manner ensuring they did not come into contact with the tank's bottom.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3940393/v1/87892930b503769513ca06e0.png"},{"id":51681447,"identity":"103b4790-b276-4a84-ad60-5a26181a75a5","added_by":"auto","created_at":"2024-02-27 06:45:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":41089,"visible":true,"origin":"","legend":"\u003cp\u003ePrediction plots comparing predicted settlement across sound treatments. Error bars represent 95 % confidence intervals of model prediction. Average settlement from each day is represented in circles. Letters represent significant differences in the treatment, same letters mean no significant difference between treatments, but different letters indicate a significant difference.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3940393/v1/7ef5c59e7786fbd4eeb14ad5.png"},{"id":51680927,"identity":"f0671fc3-af44-4c7f-aa9c-dd5b2692c3d6","added_by":"auto","created_at":"2024-02-27 06:37:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":173799,"visible":true,"origin":"","legend":"\u003cp\u003eComparison between the field and the playback spectrum levels of the experiment files\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3940393/v1/d877582e2dba62b4589434f8.png"},{"id":51681448,"identity":"e1bdba08-5879-4f4f-9cda-cafffdf7d9f5","added_by":"auto","created_at":"2024-02-27 06:45:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":65031,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the obtained acoustic metrics for the experiment and the field 1 h files. NS=no sound, OFF=off reef, R=Reef, R+V=reef and vessel, V=vessel. The definition and computation of each of the features is explained in Table S3.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3940393/v1/a08b0f2125f723d7b25c9579.png"},{"id":51682168,"identity":"abecb1ae-e9f5-4c86-adfd-8edd793ab2eb","added_by":"auto","created_at":"2024-02-27 06:53:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":704813,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3940393/v1/f203e55d-6504-42d6-b216-d7374eea1092.pdf"},{"id":51680926,"identity":"1a853644-863b-425d-a6e4-9f9d4e1f7be3","added_by":"auto","created_at":"2024-02-27 06:37:11","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":123260,"visible":true,"origin":"","legend":"","description":"","filename":"NavigatingSoundscapesSupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3940393/v1/b7cb59e9add5a8dce9c608c8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Navigating Soundscapes: Attractant effect of reef sound on oyster settlement may be attenuated by vessel noise","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIdentifying a suitable habitat prior to permanently transitioning to a benthic life stage is critical for future survival, growth and reproduction in many marine invertebrates with planktonic larvae. These species therefore evolved the perception of a variety of environmental cues, enabling them to identify promising settlement locations\u003csup\u003e1\u003c/sup\u003e. Experimental research has shown that in some species, a single cue can induce settlement and subsequent metamorphosis\u003csup\u003e1,2,3\u003c/sup\u003e. But larvae may respond to more than one cue\u003csup\u003e1\u003c/sup\u003e and sometimes even rely on a specific combination of cues\u003csup\u003e4\u003c/sup\u003e. Cues can have chemical and physical origins, and while some types of cues require close contact with a prospective settlement location, other cues may act over larger distances to guide larvae to their preferred habitat\u003csup\u003e1,5\u003c/sup\u003e. As sound propagates relatively fast and far underwater, it serves as an efficient signal transmission medium. For marine species, specific events, such as predator presences or mating opportunities can be understood by the particular sound event associated with that predator or mate\u003csup\u003e6,7\u003c/sup\u003e. However, collectively, soundscapes can also convey the overall quality and suitability of the environment for a species\u003csup\u003e8,9\u003c/sup\u003e. Research on acoustic cues informing larvae about optimal habitats has only been established relatively recently\u003csup\u003e10,11\u003c/sup\u003e. In certain invertebrate species with a settlement/metamorphosis stage, including crabs, corals, and bivalves, acoustic cues have been shown to affect larvae swimming direction\u003csup\u003e10,11\u003c/sup\u003e, settlement propensity\u003csup\u003e12,11\u003c/sup\u003e, and the timing of metamorphosis\u003csup\u003e13,14,15\u003c/sup\u003e. In general, it seems that natural environmental sounds seem to act as a positive cue to the invertebrate species in that environment\u003csup\u003e16\u003c/sup\u003e. In coral and bivalve reefs, larvae seem to be attracted to soundscapes from healthier reefs, which produce louder and more acoustically complex sounds compared to less healthy reefs which are much quieter\u003csup\u003e12,17\u003c/sup\u003e. However, the particular characteristics of the reef soundscape (i.e. sound pressure level (SPL), specific frequencies, complex mixtures of these or other acoustic characteristics) that elicit settlement behaviors remain unclear.\u003c/p\u003e\n\u003cp\u003eAnthropogenic sounds may interfere with or mask natural marine soundscapes\u003csup\u003e8\u003c/sup\u003e. Vessel noise can mask important sound cues and consequently poorer orientation toward reef sounds in fishes\u003csup\u003e18,19\u003c/sup\u003e, and delay settlement in coral larvae (planulae)\u003csup\u003e20\u003c/sup\u003e. Anthropogenic noise can not only disrupt or reduce larval settlement but may also be (mis)interpreted as a positive cue in some taxa\u003csup\u003e14,15,21,22,23\u003c/sup\u003e. Vessel noises have been shown to increase some larvae settlement, including in the mussels \u003cem\u003ePerna canaliculus\u003c/em\u003e\u003csup\u003e14\u003c/sup\u003e and \u003cem\u003eMytilus edulis\u003c/em\u003e\u003csup\u003e22\u003c/sup\u003e. Why anthropogenic noises are interpreted as a positive cue in some taxa but are repulsive to others is unknown. The reaction to anthropogenic sounds may depend on the acoustic profile of a species\u0026apos; preferred habitat and which features of this profile are responsible for attraction\u003csup\u003e15,21,24,25\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe oviparous true oyster \u003cem\u003eMagallana gigas\u0026nbsp;\u003c/em\u003eis an important reef-building ecosystem engineer\u003csup\u003e26\u003c/sup\u003e and a valuable species for aquaculture\u003csup\u003e27\u003c/sup\u003e. But in many areas across the globe it is invasive and considered a biofouling pest\u003csup\u003e28\u003c/sup\u003e that poses a threat to local species and ecosystems\u003csup\u003e29\u003c/sup\u003e. There is considerable interest in settlement preferences of this species for both bolstering as well as reducing recruitment\u003csup\u003e30\u003c/sup\u003e. The availability of settlement cues is crucial for reef sustainment, with some reports suggesting that these cues may outweigh other recruitment factors such as local hydrodynamics, and larvae supply\u003csup\u003e31,32\u003c/sup\u003e. The recent revelation that oysters not only settle more rapidly but also exhibit horizontal swimming movements toward sound sources underscores the significance of soundscapes as a navigation tool for larvae\u003csup\u003e11\u003c/sup\u003e. So far, the larvae of \u003cem\u003eM. gigas\u003c/em\u003e have not been studied for their response to acoustic settlement cues (but see Stocks et al, (2012)\u003csup\u003e21\u003c/sup\u003e for an account on swimming activity in response to natural and vessel sounds). Other true oysters with relevant experimental data are the closely related and also oviparous \u003cem\u003eCrassostrea virginica,\u003c/em\u003e and the more distantly related larviparous \u003cem\u003eOstrea angasi\u003c/em\u003e. Experimental studies have shown that both \u003cem\u003eC. virginica\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;O. angasi\u003c/em\u003e larvae prefer louder reef sounds over more quiet off-reef playbacks or no-sound controls\u003csup\u003e11,33,34\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn this study, we present the results of laboratory-playback based settlement experiments on the role of acoustic cues in settlement and metamorphosis of \u003cem\u003eMagallana gigas\u003c/em\u003e. Firstly, we were interested in the importance of oyster reef sound compared to off-reef sound. Secondly, we wanted to know whether vessel noise attracts or repels pediveliger larvae. To do so, we exposed the larvae\u003cem\u003e\u0026nbsp;\u003c/em\u003eto different vessel and reef sounds as well as off-reef and no-sound controls. Finally, we submitted the larvae to vessel and reef sounds simultaneously in order to find out whether vessel noise modifies, or even completely masks oyster reef sound cues.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch3\u003eSoundscape measurements\u003c/h3\u003e\n\u003cp\u003eAll the recordings used during the experiment were recorded in two regions of the North Sea: the Southern Bight near the Belgian coast and in the Dutch Wadden Sea (see Figure 1). Details of the data collection are explained in the Supporting Information (Appendix S1). The recorded data were manually scrolled through to select suitable files for the off-reef and the vessel treatments. Only data from spring and summer were considered, in order to correspond to the sounds from the Wadden Sea (reef and off-reef). Selected data are listed in the Supporting Information Appendix S1, Table S2. \u0026nbsp;In total, 3 recordings of reefs from 2 different locations, 4 vessel recordings with several boats on each recording from 4 different locations, and 4 off-reef recordings from 3 different locations were used to represent our treatments.\u003c/p\u003e\n\u003ch3\u003eSound files\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eThe collected sounds were scanned to select appropriate sound snippets. These segments were selected to be representative of each treatment. For example, reef sounds were only selected when they contained no apparent outside influences (e.g. vessel sounds). For the vessel sounds, a fair variability of sounds was selected, from short sounds of distant vessels to longer continuous sounds from vessels operating close by, with no other audible background sounds. The selected segments were then combined to create one 1 h file per treatment and day. In some cases, the selection led to files shorter than 1 h, so the segments were repeated and combined by applying crossfading with Audacity\u003csup\u003e35\u003c/sup\u003e to create a 1 h file. When enough recordings were available for 1 h or more, segments were not repeated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThroughout the experiment, the treatment groups were consistent but the sound file differed in each replicate. For reef treatment, sounds used were recorded from the same location in Texel, NL but sound files used during each day of the experiment were selected from different recording dates \u0026nbsp; (see Table S2). For the off-reef sounds two sound files were used recorded from Texel, NL and two sound files were used recorded from non-reef areas in the Southern Bight off the coast of Belgium (see Table S2). All vessel sounds were recorded from locations in the Southern Bight (see Table S2). Treatments where vessel sounds and reef sounds were played together were created artificially overlaying the reef sound file and the vessel sound file. The use of multiple sound files of the same treatment was used to strengthen confidence that the sounds were representative for the overall soundscape and not for a single event.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe files used were acquired with different instruments and at different locations. To deal with the difference in sampling rate and minimum recording frequency, all the files were filtered using a butterworth bandpass filter (N=4) between 20 Hz and 12 kHz. After the filtering, all the files were downsampled or upsampled to 48 ksps to match the playback requirements. Information about the instruments used for recording can be found in Table S1.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eBroodstock and Larvae Culture\u003c/h3\u003e\n\u003cp\u003eTen mature adult oysters (five females and five males) were purchased from the Guernsey Sea Farms Ltd (Guernsey, UK) and used to produce larvae. Eggs were fertilized by gonad stripping following FAO guidelines\u003csup\u003e36\u003c/sup\u003e. Fertilized eggs were kept undisturbed in flat bottom tanks for 48 hours at 22 \u0026deg;C at a density of ten eggs per ml of filtered seawater (FSW). All seawater used in this experiment was filtered at 0.1 \u0026micro;m and passed through UV light. After 48 hours larvae were sieved over 70 \u0026micro;l nylon mesh, rinsed, and transferred to rearing tanks with FSW. Tanks were aerated and kept at 22 \u0026deg;C for the entire duration of larvae rearing. Every two days larvae were sieved over mesh corresponding to the average size of the larvae and the water in the tanks was changed. Larvae were fed a mixture of fresh microalgae mixture consisting of Chaetoceros muelleri, and Isochrysis galbana (clone T-ISO). For the first 4 days larvae were fed at 40,000 cells/ml water using only I. galbana (clone T-ISO). Days 5-12 larvae were fed C. muelleri, and I. galbana (clone T-ISO) at 100,000 cells/ml at a volume ratio of 1:1. Days 13+ larvae were fed C. muelleri, and I. galbana (clone T-ISO) at 100,000 cells/ml at a volume ratio of 3:1. Larvae entered their pediveliger stage and became competent to settle at 29 days. Larvae were determined for competence when they had a prominently displayed eyespot and larval foot and were sized at 320-350 \u0026mu;m in diameter.\u003c/p\u003e\n\u003ch3\u003eSettlement Experiment Design\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eThe experiment consisted of five sound treatments: oyster reef sounds, vessel noise, reef sounds with added vessel noise, off-reef sounds, and a no-sound control. Larvae were exposed to each of these treatments in parallel, with trials that lasted 24 hours, these trials were replicated four times over four days. On each day, larvae were assessed for settlement and then discarded. At the start of each experiment day, new pediveliger stage larvae were used. Sound treatments took place in separate tanks. In each tank, 5 jars each containing 10 larvae, were used as subreplicates (see Figure 2).\u003c/p\u003e\n\u003ch4\u003eTank set-up\u003c/h4\u003e\n\u003cp\u003eFive 100L tanks were used (49x65x50.5 cm), separated 20 cm from each other on a rack. Each tank sat upon a 4 cm layer of polystyrene to isolate it from the rack and an additional layer of acoustically absorbent foam (25mm thick) between the polystyrene and the tank bottom. The acoustic foam was also placed at the tank sides. Four Lubell UW30 Underwater Speakers with custom-made amplifiers, battery-powered in order to avoid 50 Hz noise, were used. Each speaker was connected to one TASCAM playback device which played on repeat a 1 to 2 h file. No speaker was placed in the no-treatment control (see Figure 2). The speakers were hung in the middle of the tank with ropes so they would not touch the tank walls. Larvae were placed inside 100 ml polystyrene jars and these containers were fixed in the same position in the tank for every day of the experiment.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eSettlement Assays\u0026nbsp;\u003c/h4\u003e\n\u003cp\u003eOyster larvae were reared in a laboratory scale hatchery in the same facilities as where the experiments were conducted. A detailed account on the larviculture can be found in the Supporting Information.\u003c/p\u003e\n\u003cp\u003eOn each day, 10 larvae were gently pipetted randomly into each of the five 100 ml containers per tank and filled with filtered seawater (FSW) and 0.2 grams of oyster shells which could act as a settlement substrate. To get a consistent shell topography, shells were crushed using a hammer and crushed shells were sieved between 1.0 mm and 0.5 mm metal sieve. For each treatment tank, 5 individual containers were used. As all treatments were repeated over 4 consecutive days, 20 jars were used per treatment in total. All trials were conducted in a dark environment at 20 (\u0026plusmn;1) \u0026deg;C in a climate-controlled room.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo avoid any air in cups containing larvae, larvae were placed in the cups and the lid was fixed while the cup was fully submerged in FSW. This step was necessary to prevent any distortion of the sounds due to reflection from air bubbles. All FSW used in the experiment had added microalgae \u003cem\u003eChaetoceros muelleri\u003c/em\u003e, and \u003cem\u003eIsochrysis galbana\u003c/em\u003e (clone T-ISO) at 100,000 cells/ml at a volume ratio of 3:1. In a previous study, \u003cem\u003eM. gigas\u003c/em\u003e larvae increased swimming when exposed to reef sounds, but only if larvae were fed\u003csup\u003e21\u003c/sup\u003e, thus microalgae were added to our larvae containers. Microalgae were added at the same concentration as used in larvae rearing tanks and food levels were not limiting for the duration of the experiment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn top of each tank, cups were attached to a wooden pole sitting horizontally across the tank. Each larvae jar was attached so that it was in a fixed position for the duration of the experiment, the position of the jar was noted so that the effect from placement in the tank could be ruled out. The wooden pole was isolated from the tank walls with polystyrene to avoid vibration propagation. One of the cups was located directly above the speaker and the other 4 cups were at the same distance from the center of the speaker (see Figure 2).\u003c/p\u003e\n\u003cp\u003eAfter 24 hours of exposure, larvae metamorphosis was checked using a dissecting microscope and the number of larvae that had cemented themselves to the substrates were counted. Metamorphosis was confirmed by gently blowing water over the larvae with a pipette to ensure that larvae were fixed to the substrate.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003ePlayback and sound characteristics\u003c/h3\u003e\n\u003cp\u003eFor each treatment, a playback volume was chosen so the exposure power spectral density (PSD) would match the sound levels specified in literature as typical of reefs (at 1 m from the seafloor) and off-reefs (at 2 km from the reef)\u003csup\u003e11,37\u003c/sup\u003e. Details of the process done to achieve this are specified in Supporting Information.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo quantify the exposure sound level and the acoustic characteristics of each playback, each treatment was recorded using the chosen playback volume for 1 h (experiment files) at 48 ksps. When recording these 1 h files, all four different sound treatments of that batch were on to record possible acoustic crosstalk from the other treatments. These 1 h recordings were used to compute the exposure acoustic metrics for each treatment. The no-sound treatment was also recorded while all the other treatments were on. Furthermore, the room noise was also recorded using the same protocol when no speaker was active.\u003c/p\u003e\n\u003cp\u003eFor each treatment, several acoustic features were computed for both the 1 h experiment files and the 1 h field files. Acoustic Complexity Index (ACI), Acoustic Evenness Index (AEI) and Acoustic Diversity Index (ADI) were computed using the maad python package\u003csup\u003e38\u003c/sup\u003e, and the Power Spectrum Density (PSD) was computed using the scipy python package\u003csup\u003e39\u003c/sup\u003e. The average PSD was computed for three different bands by averaging the spectrum density of all the frequency bins included in the specified frequency band. Parameters used to compute each of the features are summarized in Table S3.\u003c/p\u003e\n\u003cp\u003eBoth ACI and ADI are proxies to quantify acoustic complexity (the higher the number, the more complex), while low values of AEI represent an even sound and higher values represent more uneven sounds. This is not correlated with the ecological concept of evenness, as acoustic evenness refers to an even distribution of sound energy in different frequency bands, and this can be achieved due to a high biodiversity vocalizing at the same time covering all the frequency bands or by constant broadband sounds such as some anthropogenic sounds\u003csup\u003e40,41\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eStatistical analyses\u003c/h4\u003e\n\u003cp\u003eA generalized linear mixed-effect model was created using the glmer function of the lmer package\u003csup\u003e42\u003c/sup\u003e in R version 4.1.3 (2022-03-10) (R Core Team, 2021). As the binary response variable was binary (settled vs. not settled) we fitted a Bernoulli distribution using a logit link function. The assumptions of the model were met. N value was between 175 and 180 for each treatment. The sound treatment was the only main effect variable. Individual speaker-playback, the tank used, the position of the \u0026lsquo;larvae container\u0026rsquo; within the tank, and the date of the trial (as a factor) were added as main effects to the generalized linear models to determine whether these confounders had any effect on settlement. As there was no significant main effect of speaker, tank, or cup position, it was assumed that they did not have an effect on the experiment outcome and were not included in the final model. The effect of date of the experiment was significant and therefore included as a random effect variable in the final model. Post hoc tests were performed using the emmeans function of the lsmeans package\u003csup\u003e43\u003c/sup\u003e to calculate the marginal means adjusting p-values for multiple comparisons with Tukey\u0026apos;s method and the pairs function was used to display pairwise comparisons. Post hoc analyses were also conducted on models where experiment day was included as a main effect variable in order to ensure that this variable did not interfere with the treatment.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eSettlement rate\u003c/p\u003e \u003cp\u003eLarvae settled increased 1.40 times in response to reef sound compared to vessel sounds (β\u0026thinsp;=\u0026thinsp;0.720, SE\u0026thinsp;=\u0026thinsp;0.217, p\u0026thinsp;=\u0026thinsp;0.011), 1.41 times compared to off-reef sounds (β\u0026thinsp;=\u0026thinsp;0.710, SE\u0026thinsp;=\u0026thinsp;0.218, p\u0026thinsp;=\u0026thinsp;0.010), and 1.65 times compared to the no sound treatment (β\u0026thinsp;=\u0026thinsp;0.971, SE\u0026thinsp;=\u0026thinsp;0.219, p\u0026thinsp;=\u0026thinsp;0.0001; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). When vessel sound was added to the reef sound, the settlement propensity decreased 1.28 times compared to the pure reef sound (β\u0026thinsp;=\u0026thinsp;0.540, SE\u0026thinsp;=\u0026thinsp;0.215, p\u0026thinsp;=\u0026thinsp;0.088), and was 1.10 times higher than in the vessel-only sound treatment (β\u0026thinsp;=\u0026thinsp;0.162, SE\u0026thinsp;=\u0026thinsp;0.218, p\u0026thinsp;=\u0026thinsp;0.946). Comparisons among other treatments revealed only minor differences (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Vessels and off-reef sounds had very similar effects on settlement. The lowest settlement rates were observed in a no-sound control treatment. Model predictions are plotted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe results of the posthoc of the GLMER model 1 using all data and comparing all treatments. Significant values (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) are in \u003cb\u003ebold\u003c/b\u003e, while trends (p\u0026thinsp;\u0026le;\u0026thinsp;0.10 \u0026amp; \u0026gt; 0.05) are in \u003cem\u003eitalic\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eContrasting treatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEstimate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ez.ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep.value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ereef - off reef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.70952\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.0098\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ereef - vessel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.70229\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.238\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.0106\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ereef - no sound\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.97083\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.219\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.440\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ereef - (reef\u0026thinsp;+\u0026thinsp;vessel)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.54007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-2.510\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003e0.0884\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eoff reef - vessel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.00723\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eoff reef - no sound\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.26131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-1.180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.7629\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eoff reef - (reef\u0026thinsp;+\u0026thinsp;vessel)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.16945\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.776\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9375\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eno sound - vessel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.26855\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.216\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.7418\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eno sound - (reef\u0026thinsp;+\u0026thinsp;vessel)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.43076\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.219\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2825\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003evessel - (reef\u0026thinsp;+\u0026thinsp;vessel)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.16222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.746\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9457\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePlayback\u003c/p\u003e \u003cp\u003eThe recorded sound in the tanks did not perfectly match the spectrum of the sounds recorded in the field due to the technical limitations of the reproduction equipment and the resonances that inevitably occur in tank-based experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). However, the same patterns were observed when computing for different acoustic metrics (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of our laboratory experiment showed a clear attracting effect of sounds of conspecific reefs for larvae of the oyster \u003cem\u003eMagallana gigas\u003c/em\u003e. Settlement increased about 1.41 and 1.65 times under the oyster reef treatment compared to off-reef and no sound control treatments, respectively. We further show, for the first time in any oyster species, that vessel sound might mask oyster reef sound, effectively reducing the latter\u0026rsquo;s attractive effect.\u003c/p\u003e \u003cp\u003ePreference for oyster reef sounds\u003c/p\u003e \u003cp\u003eLarvae of \u003cem\u003eM. gigas\u003c/em\u003e consistently settle more readily when exposed to sounds of reefs inhabited by conspecifics. Our finding thus corroborates earlier research in fish, corals, and other oyster species, where larvae were found to increase settlement or orient more readily towards playback of reef sounds\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Yet the sound features that trigger this response still remain to be identified. In general, oyster and coral reefs exhibit higher levels of sound and greater acoustic diversity than off-reef counterparts, due to increased soniferous biological activity including vocalizations of soniferous fishes and invertebrates, both passive or active, as well as the physical complexity of the reef\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. It remains undecided in the literature if larvae are able to distinguish particular sounds from different habitats, or if there is simply a preference for certain acoustic features such as SPL\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The spectrum of reef sounds recorded for our study followed patterns similar to other oyster reefs\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, and were half the time louder, had consistently a higher acoustic complexity, and higher evenness (lower AEI value) than off-reef areas. Compared to the vessel sounds, however, our reef sounds tended to have similar or lower PSD (depending on the vessel). Reef sounds were unique amongst the other treatments in their diversity, with consistently higher ACI and ADI values, and lower AEI values. This indicates that loudness (SPL) alone is not responsible for larval attraction, instead spectro-temporal patterns responsible for a high ACI may play a more important role. This conclusion can be corroborated by reviewing trends in the literature related to the effect of anthropogenic noise on larvae.\u003c/p\u003e \u003cp\u003ePine et al., (2016)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e found similarly that spectral composition rather than SPL is more relevant in the attraction of crab megalopae to native habitat sounds, as crab megalopae reduce metamorphosis (in comparison to natural habitat sounds) when exposed to wind turbine noise but when the same turbine noises were played back at higher SPL, this did not result in any further changes to crab metamorphosis time. For scallop larvae, Gigot et al., (2023)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e found different responses from two sources of anthropogenic noise, larvae reduced metamorphosis during drilling sounds but increased when exposed to pile driving sounds. As both sounds were substantially louder than the no sound control, this further indicates the importance of temporal and spectral composition over preference for loud sounds. Preference for louder sounds should not be ruled out completely, Wilkens et al. (2012)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e found that when exposed to (the same) vessel sound at increasingly louder SPLs, mussel larvae increased settling at the louder treatments. Lillis et al., (2016)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e also conclude that louder reefs attract more coral settlers than quieter reefs. It is reasonable to conclude that both of these sound qualities (loudness and spectro-temporal patterns) are perceptible to larvae, the preferences for each seem to be highly species-specific and could be based on the preferred habit qualities.\u003c/p\u003e \u003cp\u003eSound masking by vessel noise\u003c/p\u003e \u003cp\u003eWe found that the addition of vessel noise to the oyster reef sound led to reduced settlement in \u003cem\u003eM. gigas\u003c/em\u003e. This finding could be interpreted in one of two ways. Vessel sounds could be masking the attraction-effects of the reef sounds. Alternatively, vessel sounds could have some intrinsically repulsive component for larvae. Given that vessel sounds alone do not reduce settlement compared to sounds from off-reef areas, acoustic masking of preferred habitat sounds seems to be the more parsimonious explanation. Holles et al. (2013)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e similarly found that fish larvae will avoid swimming toward reef and vessel sounds together more often compared to reef sounds, but did not consistently avoid vessel sounds alone more than the no sound control. They concluded that the larvae might have already experienced boat noise in the ocean and become conditioned to it, or simply because boat noise in the ocean does not represent a need to change their behavioral responses. However, in coral planulae vessel noises do not only mask habitat sounds but also independently produce a negative reaction. Vessel sounds decrease the attraction of coral larvae planulae to chemical cues associated with their preferred habitats\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In comparison to other oyster species, our results corroborate well with the results from McAfee et al. (2022)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, who found that enrichment of experimental oyster reefs with reef sound in the field was ineffective in an area with high anthropogenic background noise. Future works should confirm the responses of oysters seen here, under vessel noises, including adding vessels of different origins and ranges of spectrum level (SPL). Regardless if larvae are disturbed by anthropogenic sounds by masking or direct avoidance of the sound, these disruptions to settlement, force larvae to increase their pelagic period, resulting in increased predation risk or transport to non-ideal locations.\u003c/p\u003e \u003cp\u003eThe masking effect from vessels is only established in our study with a non-significant trend (p\u0026thinsp;=\u0026thinsp;0.08). The effect size, however, is substantial with larvae being 1.28 times less likely to settle when vessel noise is added to the oyster reef sound, which is similar to the vessel noise - oyster reef comparison (reduction by 1.40 times). Treatments of reef and vessel noises together also did not differentiate significantly from off-reef and no sound controls. It is clear that the addition of vessel noises changed the characteristics of the reef sounds in our recorded files. We observed a drastic decrease of ACI, a mild decrease of ADI, and an increase of AEI, when vessel noises were added to reefs. Therefore, even though larval reaction remains uncertain, this shows that vessel noise masks the complexity of reef sounds, possibly in a way that larvae cannot distinguish them anymore.\u003c/p\u003e \u003cp\u003eSound detection by larvae\u003c/p\u003e \u003cp\u003eAquatic invertebrates, including oysters, sense particle motion rather than sound pressure\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. However, particle motion currently remains challenging to quantify, especially in small tanks. In the free field, sound pressure and particle motion are related, but that is not the case when close to reflecting and pressure relieving surfaces. Therefore, in smaller spaces such a tank, the sound propagation will not necessarily be related to particle motion because the walls and surface will act as pressure release surfaces\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Hence, sound pressure measurements can be a poor indicator of the particle motion levels, especially close to the tank walls. However, the magnitude and direction of the particle motion are expected to differ substantially from the one the larvae would experience in the field. This does not pose a problem for the current experiment, as our target was a proof of concept study into whether the settlement rate presented any differences when exposed to different acoustic stimuli in lab conditions, and to do so a fully controlled environment is necessary. Furthermore, the fluctuations over time at a large enough time scale represented in the ACI values are probably not affected by the dynamics of the tank resonances. Hence this clue remains valid. Nonetheless, future research would be necessary to confirm our results under field conditions where particle motion and sound pressure have a more direct relationship. However, in the field, it is currently possible to control added sound, but not possible to remove other background sounds.\u003c/p\u003e \u003cp\u003eImplications for reef restoration\u003c/p\u003e \u003cp\u003eDue to widespread habitat destruction, oyster reefs have been diminishing globally\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. As oysters are gregarious organisms, relying heavily on the presence of conspecifics to initiate settlement, reef destruction leads to the loss of individuals, further decreasing the chance of successful recruitment and inhibiting reef regrowth. Classic oyster reef mitigation and restoration projects focus on providing new hard substrates for wild larvae to settle, as well as supplying new adults to reefs, however, the importance of acoustic cues may be overlooked. Recently McAfee et al., (2022)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e used underwater speakers to enhance soundscapes on constructed reefs resulting in greater initial settlement of the oyster \u003cem\u003eOstrea angasi\u003c/em\u003e. If similar acoustic preferences are established for other species, these same techniques could be employed elsewhere. Our results indicate that \u003cem\u003eM. gigas\u003c/em\u003e also responds to acoustic cues, and thus may respond positively to acoustic enrichment as a restoration strategy. Our study also indicates that disruptions to natural habitat sounds from anthropogenic sources may hinder oyster settlement. The acoustic profile of an area should therefore be considered when choosing restoration sites.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe show that \u003cem\u003eM. gigas\u003c/em\u003e larvae will settle more readily in response to the sounds of oyster reefs. The reef sounds were unique in being very acoustically diverse (high ACI), while other acoustic features, such as SPL varied among treatments. This indicates that oyster larvae may be able to detect complex spectro-temporal patterns in the soundscape rather than rely solely on SPL. Furthermore, we find that noises from vessels do not repel larvae any more than the effect of off-reefs sounds or no sound controls. Rather, vessel noises seem to mask attractant reef sounds. While acoustic enrichment may bolster recruitment in oyster reefs, our results suggest that it may be less effective in areas with high levels of vessel noise pollution. We call for more research to replicate our findings in the laboratory and also to incorporate tests for sound masking by vessel noise in field experiments. More quantitative evidence is needed to estimate the critical threshold at which vessel noise may negatively affect oyster recruitment in ecologically realistic settings in the field.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eSS, CP, JH, and PH conceived the ideas and designed the methodology; MSW, ED, and CP collected the sound data; SS and CP collected the experiment data; SS and PH analyzed the data statistically; CP analyzed the acoustic data; SS and CP led the writing of the manuscript with contributions from all other co-authors.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003ePart of this project was funded by the Research Foundation Flanders (FWO) as part of the Belgian contribution to the LifeWatch ESFRI. We would like to thank the crew of the RV Simon Stevin for the help with acquiring the acoustic data. This study would have not been possible without the lending of the playback equipment from Leiden University. The authors would also like to thank Mattias Bossaer for his help with the lab set-up. Funding for the reef recordings in the Netherlands part of the Swimway Waddenzee project; financial contributions from the Waddenfund, the Ministry of Agriculture, Nature Management and Food Safety, and the Dutch provinces Noord-Holland, Groningen and Friesland.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConflicts of interest\u003c/p\u003e\n\u003cp\u003eThe authors state that there are no conflicts of interest pertaining to the research, authorship, and publication of this article. There are no financial, personal, or professional associations that might have influenced or could be perceived to have influenced the research, findings, or interpretations presented in this manuscript. The authors affirm that the content of this paper is based solely on the objective analysis and presentation of the research data.\u003c/p\u003e\n\u003cp\u003eData availability statement\u003c/p\u003e\n\u003cp\u003eAll data supporting the results in this paper are located in a Github repository. https://github.com/sschmidlin/larvae-and-sound\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHadfield, M., \u0026amp; Paul, V. Natural Chemical Cues for Settlement and Metamorphosis of Marine-Invertebrate Larvae. Marine Chemical Ecology, 431\u0026ndash;461 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurke, R. D. Pheromonal Control of Metamorphosis in the Pacific Sand Dollar, Dendraster excentricus. Science 225, 442\u0026ndash;443 (1984).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePearce, C. M. \u0026amp; Scheibling, R. E. Induction of Metamorphosis of Larvae of the Green Sea Urchin, \u003cem\u003eStrongylocentrotus droebachiensis\u003c/em\u003e, by Coralline Red Algae. The Biological Bulletin 179, 304\u0026ndash;311 (1990).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChia, F.-S. \u0026amp; Koss, R. Induction of Settlement and Metamorphosis of the Veliger Larvae of the Nudibranch, Onchidoris bilamellata. International Journal of Invertebrate Reproduction and Development 14, 53\u0026ndash;69 (1988).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcAfee, D. \u003cem\u003eet al.\u003c/em\u003e Soundscape enrichment enhances recruitment and habitat building on new oyster reef restorations. Journal of Applied Ecology 60, 111\u0026ndash;120 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmorim, M. C. P., Vasconcelos, R. O. \u0026amp; Fonseca, P. J. Fish Sounds and Mate Choice. in \u003cem\u003eSound Communication in Fishes\u003c/em\u003e (ed. Ladich, F.) 1\u0026ndash;33 (Springer, Vienna, 2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHughes, A. R., Mann, D. A. \u0026amp; Kimbro, D. L. Predatory fish sounds can alter crab foraging behaviour and influence bivalve abundance. \u003cem\u003eProceedings of the Royal Society B: Biological Sciences\u003c/em\u003e 281, 20140715 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuarte, C. M. \u003cem\u003eet al.\u003c/em\u003e The soundscape of the Anthropocene ocean. Science 371, eaba4658 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiller, P. J. O. \u003cem\u003eet al.\u003c/em\u003e Behavioral responses to predatory sounds predict sensitivity of cetaceans to anthropogenic noise within a soundscape of fear. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 119, e2114932119 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVermeij, M. J. A., Marhaver, K. L., Huijbers, C. M., Nagelkerken, I. \u0026amp; Simpson, S. D. Coral Larvae Move toward Reef Sounds. PLOS ONE 5, e10660 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams, B. R., McAfee, D., \u0026amp; Connell, S. D. Oyster larvae swim along gradients of sound. Journal of Applied Ecology, \u003cem\u003e59\u003c/em\u003e(7), 1815\u0026ndash;1824 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLillis, A., Eggleston, D. \u0026amp; Bohnenstiehl, D. Estuarine soundscapes: distinct acoustic characteristics of oyster reefs compared to soft-bottom habitats. Mar. Ecol. Prog. Ser. 505, 1\u0026ndash;17 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStanley, J. A., Radford, C. A. \u0026amp; Jeffs, A. G. Induction of settlement in crab megalopae by ambient underwater reef sound. Behav. Ecol. 21, 113\u0026ndash;120 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilkens, S. L., Stanley, J. A. \u0026amp; Jeffs, A. G. Induction of settlement in mussel (Perna canaliculus) larvae by vessel noise. Biofouling 28, 65\u0026ndash;72 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePine, M. K., Jeffs, A. G., Wang, D. \u0026amp; Radford, C. A. The potential for vessel noise to mask biologically important sounds within ecologically significant embayments. Ocean \u0026amp; Coastal Management 127, 63\u0026ndash;73 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSol\u0026eacute;, M. \u003cem\u003eet al.\u003c/em\u003e Marine invertebrates and noise. Frontiers in Marine Science 10, (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLillis, A., Bohnenstiehl, D., Peters, J. W. \u0026amp; Eggleston, D. Variation in habitat soundscape characteristics influences settlement of a reef-building coral. PeerJ 4, e2557 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolles, S., Simpson, S., Radford, A., Berten, L. \u0026amp; Lecchini, D. Boat noise disrupts orientation behaviour in a coral reef fish. Mar. Ecol. Prog. Ser. 485, 295\u0026ndash;300 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilson, L., Constantine, R., Pine, M. K., Farcas, A. \u0026amp; Radford, C. A. Impact of small boat sound on the listening space of Pempheris adspersa, Forsterygion lapillum, Alpheus richardsoni and Ovalipes catharus. Sci Rep 13, 7007 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLecchini, D. \u003cem\u003eet al.\u003c/em\u003e Boat noise prevents soundscape-based habitat selection by coral planulae. Sci Rep 8, 9283 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStocks, J. R. Response of Marine Invertebrate Larvae to Natural and Anthropogenic Sound: A Pilot Study. TOMBJ 6, 57\u0026ndash;61 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJolivet, A. \u003cem\u003eet al.\u003c/em\u003e Validation of trophic and anthropic underwater noise as settlement trigger in blue mussels. Sci Rep 6, 33829 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGigot, M. \u003cem\u003eet al.\u003c/em\u003e Pile driving and drilling underwater sounds impact the metamorphosis dynamics of Pecten maximus (L., 1758) larvae. Marine Pollution Bulletin 191, 114969 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErbe, C. Critical ratios of beluga whales (\u003cem\u003eDelphinapterus leucas\u003c/em\u003e) and masked signal duration. The Journal of the Acoustical Society of America 124, 2216\u0026ndash;2223 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, S. \u003cem\u003eet al.\u003c/em\u003e Mid- to high-frequency noise from high-speed boats and its potential impacts on humpback dolphins. The Journal of the Acoustical Society of America 138, 942\u0026ndash;952 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalles, B. \u003cem\u003eet al.\u003c/em\u003e From artificial structures to self-sustaining oyster reefs. Journal of Sea Research 108, 1\u0026ndash;9 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBotta, R., Asche, F., Borsum, J. S. \u0026amp; Camp, E. V. A review of global oyster aquaculture production and consumption. Marine Policy 117, 103952 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcAfee, D. \u0026amp; Connell, S. D. The global fall and rise of oyster reefs. Frontiers in Ecology and the Environment 19, 118\u0026ndash;125 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuesink, J. L. \u003cem\u003eet al.\u003c/em\u003e Introduction of Non-Native Oysters: Ecosystem Effects and Restoration Implications. Annual Review of Ecology, Evolution, and Systematics 36, 643\u0026ndash;689 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeck, M. W. \u003cem\u003eet al.\u003c/em\u003e Oyster Reefs at Risk and Recommendations for Conservation, Restoration, and Management. \u003cem\u003eBioScience\u003c/em\u003e 61, 107\u0026ndash;116 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePineda, J., Porri, F., Starczak, V. \u0026amp; Blythe, J. Causes of decoupling between larval supply and settlement and consequences for understanding recruitment and population connectivity. Journal of Experimental Marine Biology and Ecology 392, 9\u0026ndash;21 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoehl, M. A. R. \u0026amp; Cooper, T. Swimming in an Unsteady World. Integrative and Comparative Biology 55, 683\u0026ndash;697 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLillis, A., Eggleston, D. B. \u0026amp; Bohnenstiehl, D. R. Oyster Larvae Settle in Response to Habitat-Associated Underwater Sounds. PLOS ONE 8, e79337 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcAfee, D. \u003cem\u003eet al.\u003c/em\u003e Soundscape enrichment enhances recruitment and habitat building on new oyster reef restorations. Journal of Applied Ecology 60, 111\u0026ndash;120 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeam A. Audacity(R): Free Audio Editor and Recorder [Computer application];1999\u0026ndash;2019. (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHelm, M. M., Bourne, N., Lovatelli, A. (comp /ed), \u0026amp; Fisheries and Aquaculture Management Division. \u003cem\u003eThe Hatchery Culture of Bivalves: A Practical Manual\u003c/em\u003e. (FAO, Rome, Italy, 2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLillis, A., Bohnenstiehl, D. R. \u0026amp; Eggleston, D. B. Soundscape manipulation enhances larval recruitment of a reef-building mollusk. PeerJ 3, e999 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUlloa, J. S., Haupert, S., Latorre, J. F., Aubin, T. \u0026amp; Sueur, J. scikit-maad: An open-source and modular toolbox for quantitative soundscape analysis in Python. Methods in Ecology and Evolution 12, 2334\u0026ndash;2340 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVirtanen, P. \u003cem\u003eet al.\u003c/em\u003e SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat Methods 17, 261\u0026ndash;272 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBradfer-Lawrence, T. \u003cem\u003eet al.\u003c/em\u003e Guidelines for the use of acoustic indices in environmental research. Methods in Ecology and Evolution 10, 1796\u0026ndash;1807 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMammides, C., Goodale, E., Dayananda, S. K., Luo, K., \u0026amp; Chen, J. On the use of the acoustic evenness index to monitor biodiversity: A comment on \u0026ldquo;Rapid assessment of avian species richness and abundance using acoustic indices\u0026rdquo; by Bradfer-Lawrence et al. (2020) [Ecological Indicators, 115, 106400]. \u003cem\u003eEcological Indicators\u003c/em\u003e, \u003cem\u003e126\u003c/em\u003e, 107626. (2021)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBates, D., M\u0026auml;chler, M., Bolker, B. \u0026amp; Walker, S. Fitting Linear Mixed-Effects Models Using lme4. Journal of Statistical Software 67, 1\u0026ndash;48 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLenth, R. V. Least-Squares Means: The R Package lsmeans. Journal of Statistical Software 69, 1\u0026ndash;33 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontgomery, J. C., Jeffs, A., Simpson, S. D., Meekan, M. \u0026amp; Tindle, C. Sound as an Orientation Cue for the Pelagic Larvae of Reef Fishes and Decapod Crustaceans. in \u003cem\u003eAdvances in Marine Biology\u003c/em\u003e vol. 51 143\u0026ndash;196 (Academic Press, 2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRadford, C. A., Stanley, J. A., Simpson, S. D. \u0026amp; Jeffs, A. G. Juvenile coral reef fish use sound to locate habitats. Coral Reefs 30, 295\u0026ndash;305 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRogers, Peter H., et al. \"Parvulescu revisited: small tank acoustics for bioacousticians.\" \u003cem\u003eThe effects of noise on aquatic life II\u003c/em\u003e. Springer New York, (2016).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Larvae settlement, underwater noise, noise pollution, soundscapes, settlement cue, oyster reef ecology","lastPublishedDoi":"10.21203/rs.3.rs-3940393/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3940393/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSettlement is a critical period in the life cycle of benthic species with planktonic larval stages and for reef building invertebrates such as oysters and corals; settlement rates are predictive for reef restoration and long-term survival. Increasing evidence suggests that marine invertebrates use information from ocean soundscapes to inform settlement decisions. Sessile marine invertebrates with a settlement stage are particularly reliant on environmental cues to direct them to ideal habitats as settlement location is permanent. As gregarious settlers, oysters prefer to settle amongst members of the same species. It has been hypothesized that planktonic larvae use distinct oyster reef sounds to navigate to ideal habitats. In controlled laboratory experiments, we show that sounds recorded at conspecific reefs induce higher percentages of settlement in larvae of the Pacific Oyster \u003cem\u003eMagallana gigas\u003c/em\u003e. Additionally, we exposed larvae to anthropogenic sounds from several different vessels, combined reef-vessel sounds as well as off-reef and no sound controls. Our results suggest that attractive reef noises may be masked by vessel sounds, however, this observation is substantiated by a nonsignificant trend. Examining the acoustic characteristics of the preferred reef sounds, we hypothesize that spectro-temporal patterns are the driving attractive quality in reef sounds for this species.\u003c/p\u003e","manuscriptTitle":"Navigating Soundscapes: Attractant effect of reef sound on oyster settlement may be attenuated by vessel noise","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-27 06:37:06","doi":"10.21203/rs.3.rs-3940393/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-05T02:13:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-29T21:26:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0c156e62-bbe4-44a0-9834-a3ab7c3f3673","date":"2024-03-20T14:11:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25ca3a87-dbed-4593-9fb6-a9977bb9fe27","date":"2024-03-01T07:28:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-01T07:11:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-28T12:29:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-02-25T12:22:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-25T12:21:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-02-08T15:28:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d3f847d8-5594-467f-951c-010a0e05e5c7","owner":[],"postedDate":"February 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":28957033,"name":"Biological sciences/Ecology/Behavioural ecology"},{"id":28957034,"name":"Biological sciences/Ecology/Restoration ecology"},{"id":28957035,"name":"Physical sciences/Physics/Applied physics/Acoustics"},{"id":28957036,"name":"Earth and environmental sciences/Ocean sciences/Marine biology"}],"tags":[],"updatedAt":"2024-05-28T04:06:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-27 06:37:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3940393","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3940393","identity":"rs-3940393","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}