Acoustic fish community in the Madeira Archipelago (North Atlantic Ocean): Characterization of sound diversity and daily patterns.

Marine ecosystems are increasingly subjected to anthropogenic pressures, which demands urgent monitoring plans. Understanding soundscapes can offer unique insights into the ocean status providing important information and revealing different sounds and their sources. Fishes can be prominent soundscape contributors, making passive acoustic monitoring (PAM) a potential tool to detect the presence of vocal fish species and to monitor changes in biodiversity. The major goal of this research was to provide a first reference of the marine soundscapes of the Madeira Archipelago focusing on fish sounds, as a basis for a long-term PAM program. Based on the literature, 102 potentially vocal and 35 vocal fish species were identified. Additionally 43 putative fish sound types were detected in audio recordings from two marine protected areas (MPAs) in the Archipelago: the Garajau MPA and the Desertas MPA. The Garajau MPA exhibited higher fish vocal activity, a greater variety of putative fish sound types and higher fish sound diversity. Lower abundance of sounds was found at night at both MPAs. Acoustic activity revealed a clear distinction between diurnal and nocturnal fish groups and demonstrated daily patterns of fish sound activity, suggesting temporal and spectral partitioning of the acoustic space. Pomacentridae species were proposed as candidates for some of the dominant sound types detected during the day, while scorpionfishes (Scorpaena spp.) were proposed as sources for some of the dominant nocturnal fish sounds. This study provides an important baseline about this community acoustic behaviour and is a valuable steppingstone for future non-invasive and cost-effective monitoring programs in Madeira.

Characterization of the vocal behavior of the miniature and transparent fish model, Danionella cerebruma).

Danionella cerebrum has recently been proposed as a promising model to investigate the structure and function of the adult vertebrate brain, including the development of vocal-auditory neural pathways. This genetically tractable and transparent cypriniform is highly vocal, but limited information is available on its acoustic behavior and underlying biological function. Our main goal was to characterize the acoustic repertoire and diel variation in sound production of D. cerebrum, as well as to investigate the relationship between vocal behavior and reproduction. Sound recordings demonstrated high vocal activity, with sounds varying from short sequences of pulses known as "bursts" (comprising up to 15 pulses) to notably longer sounds, termed "long bursts", which extended up to 349 pulses with over 2.7 s. Vocal activity peaked at midday and it was very low at night with only a few bursts. While the number of pulses was higher during the daytime, the interpulse interval was longer at night. In addition, calling time was positively associated with the number of viable eggs, suggesting that acoustic communication is important for reproduction. These preliminary findings reveal the potential of using D. cerebrum to investigate vocal plasticity and the implications for sexual selection and reproduction in a novel vertebrate model for neuroscience.

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.].

The role of acoustic signals in fish reproductiona).

This paper outlines my research path over three decades while providing a review on the role of fish sounds in mate choice and reproduction. It also intends to provide advice to young scientists and point toward future avenues in this field of research. An overview of studies on different fish model species shows that male mating acoustic signals can inform females and male competitors about their size (dominant frequency, amplitude, and sound pulse rate modulation), body condition (calling activity and sound pulse rate), and readiness to mate (calling rate, number of pulses in a sound). At least in species with parental care, such as toadfishes, gobies, and pomacentrids, calling activity seems to be the main driver of reproductive success. Playback experiments ran on a restricted number of species consistently revealed that females prefer vocal to silent males and select for higher calling rates. This personal synthesis concludes with the suggestion to increase knowledge on fish mating signals, especially considering the emerging use of fish sounds to monitor aquatic environments due to increasing threats, like noise pollution.

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.

Mating sounds in the two-spotted goby, Pomatoschistus flavescens: Effects of water temperature on acoustic featuresa).

Acoustic signals in teleost fishes play a fundamental role in reproduction. As fish are ectothermic animals, temperature has the potential to change their signal production and detection, with further implications for mating interactions. In this study, we describe the mating sounds made by the two-spotted goby, Pomatoschistus flavescens, for the first time and further investigate the effect of temperature on the acoustic features. Courtship sounds of 15 two-spotted goby males were recorded at three different temperatures: 16 °C, 19 °C, and 21 °C. As seen for other marine gobies, two-spotted goby produced two courtship sounds: drums and thumps. Drums showed similar acoustic features to other Pomatoschistus species already studied. Calling rates for both kinds of sound were not affected by the increases in temperature. However, pulse rate increased from 16 °C to 19 °C and stabilised between 19 °C and 21 °C, suggesting that two-spotted gobies reached their physiological limits at 19 °C. Spectral features were also affected by temperature, presenting higher values at 19 °C. Whether or not the observed changes in acoustic features with temperature lead to changes in mating remains to be addressed. Studies like the present one are fundamental to better comprehend how reproduction will be affected by global warming in soniferous fishes.

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.

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 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.

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.

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.

Continuous but not intermittent noise has a negative impact on mating success in a marine fish with paternal care.

Anthropogenic underwater noise is a global pollutant of increasing concern but its impact on reproduction in fish is largely unknown. Hence, a better understanding of its consequences for this important link to fitness is crucial. Working in aquaria, we experimentally tested the impact of broadband noise exposure (added either continuously or intermittently), compared to a control, on the behaviour and reproductive success of the common goby (Pomatoschistus microps), a vocal fish with exclusive paternal care. Compared to the intermittent noise and control treatments, the continuous noise treatment increased latency to female nest inspection and spawning and decreased spawning probability. In contrast, many other female and male pre-spawning behaviours, and female ventilation rate (proxies for stress levels) did not differ among treatments. Therefore, it is likely that female spawning decisions were delayed by a reduced ability to assess male acoustic signals, rather than due to stress per se and that the silent periods in the intermittent noise treatment provided a respite where the females could assess the males. Taken together, we show that noise (of similar frequency range as anthropogenic boat noise) negatively affects reproductive success, particularly under a continuous noise exposure.

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.

Appraisal of unimodal cues during agonistic interactions in Maylandia zebra.

Communication is essential during social interactions including animal conflicts and it is often a complex process involving multiple sensory channels or modalities. To better understand how different modalities interact during communication, it is fundamental to study the behavioural responses to both the composite multimodal signal and each unimodal component with adequate experimental protocols. Here we test how an African cichlid, which communicates with multiple senses, responds to different sensory stimuli in a social relevant scenario. We tested Maylandia zebra males with isolated chemical (urine or holding water coming both from dominant males), visual (real opponent or video playback) and acoustic (agonistic sounds) cues during agonistic interactions. We showed that (1) these fish relied mostly on the visual modality, showing increased aggressiveness in response to the sight of a real contestant but no responses to urine or agonistic sounds presented separately, (2) video playback in our study did not appear appropriate to test the visual modality and needs more technical prospecting, (3) holding water provoked territorial behaviours and seems to be promising for the investigation into the role of the chemical channel in this species. Our findings suggest that unimodal signals are non-redundant but how different sensory modalities interplay during communication remains largely unknown in fish.

Ontogenetic development of the inner ear saccule and utricle in the Lusitanian toadfish: Potential implications for auditory sensitivity.

Studies addressing structure-function relationships of the fish auditory system during development are sparse compared to other taxa. The Batrachoididae has become an important group to investigate mechanisms of auditory plasticity and evolution of auditory-vocal systems. A recent study reported ontogenetic improvements in the inner ear saccule sensitivity of the Lusitanian toadfish, Halobatrachus didactylus, but whether this results from changes in the sensory morphology remains unknown. We investigated how the macula and organization of auditory receptors in the saccule and utricle change during growth in this species. Inner ear sensory epithelia were removed from the end organs of previously PFA-fixed specimens, from non-vocal posthatch fry (<1.4 cm, standard length) to adults (>23 cm). Epithelia were phalloidin-stained and analysed for area, shape, number and orientation patterns of hair cells (HC), and number and size of saccular supporting cells (SC). Saccular macula area expanded 41x in total, and significantly more (relative to body length) among vocal juveniles (2.3-2.9 cm). Saccular HC number increased 25x but HC density decreased, suggesting that HC addition is slower relative to epithelial growth. While SC density decreased, SC apical area increased, contributing to the epithelial expansion. The utricule revealed increased HC density (striolar region) and less epithelial expansion (5x) with growth, contrasting with the saccule that may have a different developmental pattern due to its larger size and main auditory functions. Both macula shape and HC orientation patterns were already established in the posthatch fry and retained throughout growth in both end organs. We suggest that previously reported ontogenetic improvements in saccular sensitivity might be associated with changes in HC number (not density), size and/or molecular mechanisms controlling HC sensitivity. This is one of the first studies investigating the ontogenetic development of the saccule and utricle in a vocal fish and how it potentially relates to auditory enhancement for acoustic communication.