Correction: Vocal repertoire and consistency of call features in the meagre Argyrosomous regius (Asso, 1801).

[This corrects the article DOI: 10.1371/journal.pone.0241792.].

Endemic fish calling: Acoustics and reproductive behaviour of the Neretva dwarf goby Orsinigobius croaticus.

The Neretva dwarf goby Orsinigobius croaticus (Gobiiformes, Gobionellidae) is an endemic fish native to the freshwaters of the Adriatic Basin in Croatia and Bosnia and Herzegovina, a Mediterranean Biodiversity Hotspot. Due to its limited distribution range, specific karst habitat and endangered status, laboratory studies on reproductive biology are scarce but crucial. Herein, we investigated the sound production and acoustic behaviour of the endangered O. croaticus during reproductive intersexual laboratory encounters, utilising an interdisciplinary approach. We also performed dissections and micro-computed tomography (µCT) scanning of the pectoral girdle to explore its potential involvement in sound production. Finally, comparative acoustic analysis was conducted on sounds produced by previously recorded soniferous sand gobies to investigate whether acoustic features are species-specific. The endemic O. croaticus is a soniferous species. Males of this species emit pulsatile sounds composed of a variable number of short (~15 ms) consecutive pulses when interacting with females, usually during the pre-spawning phase in the nest, but also during courtship outside the nest. Pulsatile sounds were low-frequency and short pulse trains (~140 Hz, <1000 ms). Male visual behaviour rate was higher when co-occurring with sounds and females entered the male's nest significantly more frequently when sounds were present. Characteristic body movements accompanied male sound production, such as head thrust and fin spreading. Furthermore, µCT scans and dissections suggest that O. croaticus shares certain anatomical similarities of the pectoral girdle (i.e. osseous elements and arrangement of levator pectoralis muscles) to previously studied sand gobies that could be involved in sound production. Multivariate comparisons, using sounds produced by eight soniferous European sand gobies, effectively distinguished soniferous (and sympatric) species based on their acoustic properties. However, the discrimination success decreased when temperature-dependent features (sound duration and pulse repetition rate) were excluded from the analysis. Therefore, we suggest both spectral and temporal features are important for the acoustic differentiation of sand gobies.

Effects of temperature on acoustic and visual courtship and reproductive success in the two-spotted goby Pomatoschistus flavescens.

Fish are ectothermic and small changes in water temperature could greatly affect reproduction. The two-spotted goby is a small semi-pelagic species that uses visual and acoustic displays to mate. Here, we studied the effect of temperature (16 and 20 °C) on acoustic and visual courtship and associated reproductive success in 39 males. Temperature influenced male visual courtship performed outside the nest, but it did not influence calling rate and the number of laid eggs. Interestingly, the number of sounds (drums) was the sole predictor of spawning success. These findings suggest that exposure to different temperatures within the species' natural range affect courtship behaviour but not its reproductive success. We propose that finding the link between acoustic behaviour and reproduction in fishes offers the opportunity to monitor fish sounds both in the lab and in nature to learn how they respond to environmental changes and human impacts, namely global warming.

Detection of invasive fish species with passive acoustics: Discriminating between native and non-indigenous sciaenids.

Invasive alien species have been rising exponentially in the last decades impacting biodiversity and ecosystem functioning. The soniferous weakfish, Cynoscion regalis, is a recent invasive sciaenid species in the Iberian Peninsula and was first reported in the Tagus estuary in 2015. There is concern about its possible impacts on native species, namely the confamiliar meagre, Argyrosomus regius, as there is overlap in their feeding regime, habitat use, and breeding behaviour. Here, we characterised the sciaenid-like sounds recently recorded in the Tagus estuary and showed that they are made by weakfish as they have similar numbers of pulses and pulse periods to the sounds made by captive breeding weakfish. We further demonstrate that breeding grunts from weakfish and the native sciaenid, recorded either in captivity or Tagus estuary, differ markedly in sound duration, number of pulses and pulse period in the two species, but overlap in their spectral features. Importantly, these differences are easily detected through visual and aural inspections of the recordings, making acoustic recognition easy even for the non-trained person. We propose that passive acoustic monitoring can be a cost-effective tool for in situ mapping of weakfish outside its natural distribution and an invaluable tool for early detection and to monitor its expansion.

Impact of anthropogenic noise on the survival and development of meagre (Argyrosomus regius) early life stages.

The growth of human populations has been driving an unprecedent and widespread increase in marine traffic, posing a real threat to marine biodiversity. Even though we are now aware of the negative effects of shipping noise exposure on fish, information about the impact on their early life stages continues to lack. Meagre (Argyrosomus regius) is a vocal fish that uses estuaries with high levels of anthropogenic noise pollution as both breeding areas and nurseries. Here, the effects of boat noise exposure on the development and survival of meagre larvae were studied. Embryos and larvae were exposed to either noise (boat noise playback) or control treatments (coils producing a similar electric field to the speakers) and hatching rate, survival rate, morphometric traits and stress-related biomarkers, at hatching and at 2 days-post-hatching (dph) were analyzed. Results showed no conclusive effects of the impact of boat noise playback, even though there was an increased lipid droplet consumption and a decrease in body depth at 2dph larvae under this stressor. The assessment of oxidative stress and energy metabolism-related biomarkers at hatching showed a marginal decrease in superoxide dismutase (SOD) activity and no changes in DNA damage or electron transport system activity (ETS), although it cannot be disregarded that those effects could only be visible at later stages of larval development. Whether these morphological and developmental results have implications in later stages remains to be investigated. Further studies with longer exposure and wild meagre could help deepen this knowledge and provide a better understanding of how anthropogenic noise can impact meagre early stages.

Boat noise impacts Lusitanian toadfish breeding males and reproductive outcome.

Anthropogenic noise is a growing threat to marine organisms, including fish. Yet very few studies have addressed the impact of anthropogenic noise on fish reproduction, especially in situ. In this study, we investigated the impacts of boat noise exposure in the reproductive success of wild Lusitanian toadfish (Halobatrachus didactylus), a species that relies on advertisement calls for mate attraction, using behavioural, physiological and reproductive endpoints. Two sets of artificial nests were deployed in the Tagus estuary and exposed to either ambient sound or boat noise during their breeding season. Toadfish males spontaneously used these nests to breed. We inspected nests for occupation and the presence of eggs in six spring low tides (in two years) and assessed male vocal activity and stress responses. Boat noise did not affect nest occupation by males but impacted reproductive success by decreasing the likelihood of receiving eggs, decreasing the number of live eggs and increasing the number of dead eggs, compared to control males. Treatment males also showed depressed vocal activity and slightly higher cortisol levels. The assessment of oxidative stress and energy metabolism-related biomarkers revealed no oxidative damage in noise exposed males despite having lower antioxidant responses and pointed towards a decrease in the activity levels of energy metabolism-related biomarkers. These results suggest that males exposed to boat noise depressed their metabolism and their activity (such as parental care and mate attraction) to cope with an acoustic stressor, consistent with a freezing defensive response/behaviour. Together, our study demonstrates that boat noise has severe impacts on reproductive fitness in Lusitanian toadfish. We argue that, at least fishes that cannot easily avoid noise sources due to their dependence on specific spawning sites, may incur in significant direct fitness costs due to chronic noise exposure.

Noise-induced hearing loss correlates with inner ear hair cell decrease in larval zebrafish.

Anthropogenic noise can be hazardous for the auditory system and wellbeing of animals, including humans. However, very limited information is known on how this global environmental pollutant affects auditory function and inner ear sensory receptors in early ontogeny. The zebrafish (Danio rerio) is a valuable model in hearing research, including investigations of developmental processes of the vertebrate inner ear. We tested the effects of chronic exposure to white noise in larval zebrafish on inner ear saccular sensitivity and morphology at 3 and 5 days post-fertilization (dpf), as well as on auditory-evoked swimming responses using the prepulse inhibition (PPI) paradigm at 5 dpf. Noise-exposed larvae showed a significant increase in microphonic potential thresholds at low frequencies, 100 and 200 Hz, while the PPI revealed a hypersensitization effect and a similar threshold shift at 200 Hz. Auditory sensitivity changes were accompanied by a decrease in saccular hair cell number and epithelium area. In aggregate, the results reveal noise-induced effects on inner ear structure-function in a larval fish paralleled by a decrease in auditory-evoked sensorimotor responses. More broadly, this study highlights the importance of investigating the impact of environmental noise on early development of sensory and behavioural responsiveness to acoustic stimuli.

Boat noise affects meagre (Argyrosomus regius) hearing and vocal behaviour.

Aquatic noise has increased in last decades imposing new constraints on aquatic animals' acoustic communication. Meagre (Argyrosomus regius) produce loud choruses during the breeding season, likely facilitating aggregations and mating, and are thus amenable to being impacted by anthropogenic noise. We assessed the impact of boat noise on this species acoustic communication by: evaluating possible masking effects of boat noise on hearing using Auditory Evoked Potentials (AEP) and inspecting changes in chorus sound levels from free ranging fish upon boat passages. Our results point to a significant masking effect of anthropogenic noise since we observed a reduction of ca. 20 dB on the ability to discriminate conspecific calls when exposed to boat noise. Furthermore, we verified a reduction in chorus energy during ferryboat passages, a behavioural effect that might ultimately impact spawning. This study is one of few addressing the effects of boat noise by combining different methodologies both in the lab and with free ranging animals.

Fish sounds and boat noise are prominent soundscape contributors in an urban European estuary.

Passive acoustic monitoring is a valuable tool for non-intrusive monitoring of marine environments, also allowing the assessment of underwater noise that can negatively affect marine organisms. Here we provide for the first time, an assessment of noise levels and temporal soundscape patterns for a European estuary. We used several eco-acoustics methodologies to characterize the data collected over six weeks within May 2016 - July 2017 from Tagus estuary. Biophony was the major contributor dominated by fish vocalizations and the main driver for seasonal patterns. Maritime traffic was the major source of anthropogenic noise, with daily patterns monitored using 1584 Hz third-octave band level. This indicator avoided biophony and geophony, unlike other indicators proposed for the EU Marine Strategy Framework Directive. Furthermore, the frequency overlap between anthropophony and biophony demands precautionary actions and calls for further research. This study provides an assessment that will be useful for future monitoring and management strategies.

Boat noise interferes with Lusitanian toadfish acoustic communication.

Anthropogenic noise is considered a major underwater pollutant as increasing ocean background noise due to human activities is impacting aquatic organisms. One of the most prevalent anthropogenic sounds is boat noise. Although motorboat traffic has increased in the past few decades, its impact on the communication of fish is still poorly known. The highly vocal Lusitanian toadfish (Halobatrachus didactylus) is an excellent model to test the impact of this anthropogenic stressor as it relies on acoustic communication to attract mates. Here, we performed two experiments to test the impact of boat noise on the acoustic communication of the Lusitanian toadfish. Using the auditory evoked potential (AEP) technique, we first compared the maximum distance a fish can perceive a boatwhistle (BW), the mate attraction acoustic signal, before and after embedding it in boat noise. Noises from a small motorboat and from a ferryboat reduced the active space from a control value of 6.4-10.4 m to 2.0-2.5 m and 6.3-6.7 m, respectively. In the second experiment we monitored the acoustic behaviour of breeding males exposed to boat noise playbacks and we observed an increase in the inter-onset interval of BWs and a disruption of the usual vocal interactions between singing males. These results demonstrate that boat noise can severely reduce the acoustic active space and affect the chorusing behaviour in this species, which may have consequences in breeding success for individuals and could thus affect fitness.

Vocal repertoire and consistency of call features in the meagre Argyrosomous regius (Asso, 1801).

Passive Acoustic Monitoring (PAM) is a non-intrusive and cost-effective method capable of providing high-resolution, long-term information on the status and health of vocal populations and communities. To successfully monitor the same species over wide geographical and temporal scales, it is necessary to characterise the range of sound variability, as well as the consistency of sound features between populations. The meagre (Argyrosomus regius, Asso 1801) is an interesting case study because recent investigations suggest a wider vocal repertoire than previously described. In this study, meagre vocalizations were recorded and analysed from a variety of settings, ranging from rearing facilities to wild populations to provide a comprehensive characterisation of its vocal repertoire, while investigating the consistency of spawning sound features between populations. All sounds presented a similar acoustic structure in their basic unit (i.e. the pulse), while an important variability was found in the number of pulses; the meagre can emit sounds made of one single pulse or many pulses (up to more than 100). High level of overlap in the Principal Component Analysis made difficult to differentiate sound type clusters. Despite this, two sound types were identifiable: knocks (sounds from 1 to 3 pulses) and long grunts (sounds with more than 29 pulses). Discriminant Analysis carried out on PCA residuals showed that knock had the highest proportion of correct placement (92% of the observations correctly placed) followed by long grunts (80%). All other previously described sound types (intermediate grunt, short grunt and disturbance sounds) could not be separated and presented low levels of correct placement, suggesting that care should be taken when defining these as independent sound types. Finally, acoustic features consistency was found in meagre grunts emitted by different populations during spawning nights; statistical differences could be explained by recording settings and fish conditions. The results of this study provide important information for fostering PAM programs of wild meagre populations, while contributing to the discussion around the definition of fish sound types in vocal fish communities. Studies of this kind, which evaluate both variability and consistency of sound features, are of fundamental importance for maximising PAM efforts in the wild, at both the specific and the community level.

Noise-induced hearing loss in zebrafish: investigating structural and functional inner ear damage and recovery.

Exposure to continuous moderate noise levels is known to impair the auditory system leading to Noise-Induced Hearing Loss (NIHL) in animals including humans. The mechanism underlying noise-dependent auditory Temporary Threshold Shifts (TTS) is not fully understood. In fact, only limited information is available on vertebrates such as fishes, which share homologous inner ear structures to mammals and have the ability to regenerate hair cells. The zebrafish Danio rerio is a well-established model in hearing research providing an unmatched opportunity to investigate the molecular and physiological mechanisms of NIHL at the sensory receptor level. Here we investigated for the first time the effects of noise exposure on TTS and functional recovery in zebrafish, as well as the associated morphological damage and regeneration of the inner ear saccular hair cells. Adult specimens were exposed for 24h to white noise at various amplitudes (130, 140 and 150 dB re. 1 µPa) and their auditory sensitivity was subsequently measured with the Auditory Evoked Potential (AEP) recording technique. Sensory recovery was tested at different times post-treatment (after 3, 7 and 14 days) and compared to individuals kept under quiet lab conditions. Results revealed noise level-dependent TTS up to 33 dB and increase in response latency. Recovery of hearing function occurred within 7 days for fish exposed to 130 and 140 dB noise levels, while fish subject to 150 dB only returned to baseline thresholds after 14 days. Hearing impairment was accompanied by significant loss of hair cells only at the highest noise treatment. Full regeneration of the sensory tissue (number of hair cell receptors) occurred within 7 days, which was prior to functional recovery. We provide first baseline data of NIHL in zebrafish and validate this species as an effective vertebrate model to investigate the impact of noise exposure on the structure and function of the adult inner ear and its recovery process.

Sound production in the Meagre, Argyrosomus regius (Asso, 1801): intraspecific variability associated with size, sex and context.

BACKGROUND: Many fish taxa produce sound in voluntary and in disturbance contexts but information on the full acoustic repertoire is lacking for most species. Yet, this knowledge is critical to enable monitoring fish populations in nature through acoustic monitoring. METHODS: In this study we characterized the sounds emitted during disturbance and voluntary contexts by juvenile and adult meagre, Argyrosomus regius, in laboratory conditions. Breeding sounds produced by captive adults were also compared with meagre sounds registered in the Tagus estuary (Lisbon, Portugal) from unseen fish during the breeding season. RESULTS: The present dataset demonstrates for the first time that in this species dominant frequency is inversely related to fish size, and that sounds vary according to sex, context and age. Sounds from captive breeding adults were similar to sounds recorded in the field. DISCUSSION: Our findings indicate that A. regius sound features carry information about size, sex, age and motivation. This variability could potentially be used to identify meagre in the field and to infer about ontogenetic phase (i.e., juveniles vs. adults, and variation with size) and motivation (e.g., spawning). Future studies should confirm sex differences and ascertain the influence of water temperature on acoustic features.

Assessment of fighting ability in the vocal cichlid Metriaclima zebra in face of incongruent audiovisual information.

Information transfer between individuals typically depends on multiple sensory channels. Yet, how multi-sensory inputs shape adaptive behavioural decisions remains largely unexplored. We tested the relative importance of audio and visual sensory modalities in opponent size assessment in the vocal cichlid fish, Metriaclima zebra, by playing back mismatched agonistic sounds mimicking larger or smaller opponents during fights of size-matched males. Trials consisted in three 5-min periods: PRE (visual), PBK (acoustic+visual) and POST (visual). During PBK agonistic sounds of smaller (high frequency or low amplitude) or larger (low frequency or high amplitude) males were played back interactively. As a control, we used white noise and silence. We show that sound frequency but not amplitude affects aggression, indicating that spectral cues reliably signal fighting ability. In addition, males reacted to the contrasting audio-visual information by giving prevalence to the sensory channel signalling a larger opponent. Our results suggest that fish can compare the relevance of information provided by different sensory inputs to make behavioural decisions during fights, which ultimately contributes to their individual fitness. These findings have implications for our understanding of the role of multi-sensory inputs in shaping behavioural output during conflicts in vertebrates.

Characterization of the acoustic community of vocal fishes in the Azores.

Sounds produced by teleost fishes are an important component of marine soundscapes, making passive acoustic monitoring (PAM) an effective way to map the presence of vocal fishes with a minimal impact on ecosystems. Based on a literature review, we list the known soniferous fish species occurring in Azorean waters and compile their sounds. We also describe new fish sounds recorded in Azores seamounts. From the literature, we identified 20 vocal fish species present in Azores. We analysed long-term acoustic recordings carried out since 2008 in Condor and Princesa Alice seamounts and describe 20 new putative fish sound sequences. Although we propose candidates as the source of some vocalizations, this study puts into evidence the myriad of fish sounds lacking species identification. In addition to identifying new sound sequences, we provide the first marine fish sound library for Azores. Our acoustic library will allow to monitor soniferous fish species for conservation and management purposes.

Acoustic barriers as an acoustic deterrent for native potamodromous migratory fish species.

This study focused on the use of sound playbacks as acoustic deterrents to direct native potamodromous migratory species away from all kind of traps. The effects of two acoustic treatments, a repeated sine sweep up to 2 kHz (sweep-up stimulus) and an intermittent 140 Hz tone, were tested in three fish species native to Iberia: Salmo trutta, Pseudochondrostoma duriense and Luciobarbus bocagei. In contrast with S. trutta, the endemic cyprinids P. duriense and L. bocagei exhibited a strong repulse reaction to the frequency sweep-up sound. The 140 Hz stimulus did not seem to alter significantly the behaviour of any of the studied species. These results highlight the potential of acoustic stimuli as fish behavioural barriers and their application to in situ conservation measures of native Iberian fish populations, to protect them from hydropower dams. In addition, this study shows that acoustic deterrents can be used selectively on target species.

Acoustic communication in marine shallow waters: testing the acoustic adaptive hypothesis in sand gobies.

Acoustic communication is an important part of social behaviour of fish species that live or breed in shallow noisy waters. Previous studies have shown that some fish species exploit a quiet window in the background noise for communication. However, it remains to be examined whether hearing abilities and sound production of fish are adapted to marine habitats presenting high hydrodynamism. Here, we investigated whether the communication system of the painted (Pomatoschistus pictus) and the marbled (Pomatoschistus marmoratus) gobies is adapted to enhance sound transmission and reception in Atlantic shallow water environments. We recorded and measured the sound pressure levels of social vocalisations of both species, as well as snapshots of ambient noise of habitats characterised by different hydrodynamics. Hearing thresholds (in terms of both sound pressure and particle acceleration) and responses to conspecific signals were determined using the auditory evoked potential recording technique. We found that the peak frequency range (100-300 Hz) of acoustic signals matched the best hearing sensitivity in both species and appeared well adapted for short-range communication in Atlantic habitats. Sandy/rocky exposed beaches presented a quiet window, observable even during the breaking of moderate waves, coincident with the main sound frequencies and best hearing sensitivities of both species. Our data demonstrate that the hearing abilities of these gobies are well suited to detect conspecific sounds within typical interacting distances (a few body lengths) in Atlantic shallow waters. These findings lend support to the acoustic adaptive hypothesis, under the sensory drive framework, proposing that signals and perception systems coevolve to be effective within local environment constraints.

Acoustic Complexity of vocal fish communities: a field and controlled validation.

The Acoustic Complexity Index (ACI) is increasingly applied to the study of biodiversity in aquatic habitats. However, it remains unknown which types of acoustic information are highlighted by this index in underwater environments. This study explored the robustness of the ACI to fine variations in fish sound abundance (i.e. number of sounds) and sound diversity (i.e. number of sound types) in field recordings and controlled experiments. The ACI was found to be sensitive to variations in both sound abundance and sound diversity, making it difficult to discern between these variables. Furthermore, the ACI was strongly dependent on the settings used for its calculation (i.e. frequency and temporal resolution of the ACI algorithm, amplitude filter). Care should thus be taken when comparing ACI absolute values between studies, or between sites with site-specific characteristics (e.g. species diversity, fish vocal community composition). As the use of ecoacoustic indices presents a promising tool for the monitoring of vulnerable environments, methodological validations like those presented in this paper are of paramount importance in understanding which biologically important information can be gathered by applying acoustic indices to Passive Acoustic Monitoring data.

Noise can affect acoustic communication and subsequent spawning success in fish.

There are substantial concerns that increasing levels of anthropogenic noise in the oceans may impact aquatic animals. Noise can affect animals physically, physiologically and behaviourally, but one of the most obvious effects is interference with acoustic communication. Acoustic communication often plays a crucial role in reproductive interactions and over 800 species of fish have been found to communicate acoustically. There is very little data on whether noise affects reproduction in aquatic animals, and none in relation to acoustic communication. In this study we tested the effect of continuous noise on courtship behaviour in two closely-related marine fishes: the two-spotted goby (Gobiusculus flavescens) and the painted goby (Pomatoschistus pictus) in aquarium experiments. Both species use visual and acoustic signals during courtship. In the two-spotted goby we used a repeated-measures design testing the same individuals in the noise and the control treatment, in alternating order. For the painted goby we allowed females to spawn, precluding a repeated-measures design, but permitting a test of the effect of noise on female spawning decisions. Males of both species reduced acoustic courtship, but only painted gobies also showed less visual courtship in the noise treatment compared to the control. Female painted gobies were less likely to spawn in the noise treatment. Thus, our results provide experimental evidence for negative effects of noise on acoustic communication and spawning success. Spawning is a crucial component of reproduction. Therefore, even though laboratory results should not be extrapolated directly to field populations, our results suggest that reproductive success may be sensitive to noise pollution, potentially reducing fitness.