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.

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.

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.

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.

Assessing acoustic communication active space in the Lusitanian toadfish.

The active space of a signal is an important concept in acoustic communication as it has implications for the function and evolution of acoustic signals. However, it remains mostly unknown for fish as it has been measured in only a restricted number of species. We combined physiological and sound propagation approaches to estimate the communication range of the Lusitanian toadfish's ( ITALIC! Halobatrachus didactylus) advertisement sound, the boatwhistle (BW). We recorded BWs at different distances from vocalizing fish in a natural nesting site at ca. 2-3 m depth. We measured the representation of these increasingly attenuated BWs in the auditory pathway through the auditory evoked potential (AEP) technique. These measurements point to a communication range of between 6 and 13 m, depending on the spectral characteristics of the BW. A similar communication range (ca. 8 m) was derived from comparing sound attenuation at selected frequencies with auditory sensitivity. This is one of the few studies to combine auditory measurements with sound propagation to estimate the active space of acoustic signals in fish. We emphasize the need in future studies for estimates of active space to take informational masking into account.

How effective are acoustic signals in territorial defence in the Lusitanian toadfish?

The function of fish sounds in territorial defence, in particular its influence on the intruder's behaviour during territorial invasions, is poorly known. Breeding Lusitanian toadfish males (Halobatrachus didactylus) use sounds (boatwhistles) to defend nests from intruders. Results from a previous study suggest that boatwhistles function as a 'keep-out signal' during territorial defence. To test this hypothesis we performed territorial intrusion experiments with muted Lusitanian toadfish. Males were muted by making a cut and deflating the swimbladder (the sound-producing apparatus) under anaesthesia. Toadfish nest-holder males reacted to intruders mainly by emitting sounds (sham-operated and control groups) and less frequently with escalated bouts of fighting. When the nest-holder produced a boatwhistle, the intruder fled more frequently than expected by chance alone. Muted males experienced a higher number of intrusions than the other groups, probably because of their inability to vocalise. Together, our results show that fish acoustic signals are effective deterrents in nest/territorial intrusions, similar to bird song.

Vocal differentiation parallels development of auditory saccular sensitivity in a highly soniferous fish.

Vocal differentiation is widely documented in birds and mammals but has been poorly investigated in other vertebrates, including fish, which represent the oldest extant vertebrate group. Neural circuitry controlling vocal behaviour is thought to have evolved from conserved brain areas that originated in fish, making this taxon key to understanding the evolution and development of the vertebrate vocal-auditory systems. This study examines ontogenetic changes in the vocal repertoire and whether vocal differentiation parallels auditory development in the Lusitanian toadfish Halobatrachus didactylus (Batrachoididae). This species exhibits a complex acoustic repertoire and is vocally active during early development. Vocalisations were recorded during social interactions for four size groups (fry: <2 cm; small juveniles: 2-4 cm; large juveniles: 5-7 cm; adults >25 cm, standard length). Auditory sensitivity of juveniles and adults was determined based on evoked potentials recorded from the inner ear saccule in response to pure tones of 75-945 Hz. We show an ontogenetic increment in the vocal repertoire from simple broadband-pulsed 'grunts' that later differentiate into four distinct vocalisations, including low-frequency amplitude-modulated 'boatwhistles'. Whereas fry emitted mostly single grunts, large juveniles exhibited vocalisations similar to the adult vocal repertoire. Saccular sensitivity revealed a three-fold enhancement at most frequencies tested from small to large juveniles; however, large juveniles were similar in sensitivity to adults. We provide the first clear evidence of ontogenetic vocal differentiation in fish, as previously described for higher vertebrates. Our results suggest a parallel development between the vocal motor pathway and the peripheral auditory system for acoustic social communication in fish.

Call recognition and individual identification of fish vocalizations based on automatic speech recognition: An example with the Lusitanian toadfish.

The study of acoustic communication in animals often requires not only the recognition of species specific acoustic signals but also the identification of individual subjects, all in a complex acoustic background. Moreover, when very long recordings are to be analyzed, automatic recognition and identification processes are invaluable tools to extract the relevant biological information. A pattern recognition methodology based on hidden Markov models is presented inspired by successful results obtained in the most widely known and complex acoustical communication signal: human speech. This methodology was applied here for the first time to the detection and recognition of fish acoustic signals, specifically in a stream of round-the-clock recordings of Lusitanian toadfish (Halobatrachus didactylus) in their natural estuarine habitat. The results show that this methodology is able not only to detect the mating sounds (boatwhistles) but also to identify individual male toadfish, reaching an identification rate of ca. 95%. Moreover this method also proved to be a powerful tool to assess signal durations in large data sets. However, the system failed in recognizing other sound types.

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.

Mate preference in the painted goby: the influence of visual and acoustic courtship signals.

We tested the hypothesis that females of a small vocal marine fish with exclusive paternal care, the painted goby, prefer high parental-quality mates such as large or high-condition males. We tested the effect of male body size and male visual and acoustic courtship behaviour (playback experiments) on female mating preferences by measuring time spent near one of a two-choice stimuli. Females did not show preference for male size but preferred males that showed higher levels of courtship, a trait known to advertise condition (fat reserves). Also, time spent near the preferred male depended on male courtship effort. Playback experiments showed that when sound was combined with visual stimuli (a male confined in a small aquarium placed near each speaker), females spent more time near the male associated with courtship sound than with the control male (associated with white noise or silence). Although male visual courtship effort also affected female preference in the pre-playback period, this effect decreased during playback and disappeared in the post-playback period. Courtship sound stimuli alone did not elicit female preference in relation to a control. Taken together, the results suggest that visual and mainly acoustic courtship displays are subject to mate preference and may advertise parental quality in this species. Our results indicate that visual and acoustic signals interplay in a complex fashion and highlight the need to examine how different sensory modalities affect mating preferences in fish and other vertebrates.

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.

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.].

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.

Representation of complex vocalizations in the Lusitanian toadfish auditory system: evidence of fine temporal, frequency and amplitude discrimination.

Many fishes rely on their auditory skills to interpret crucial information about predators and prey, and to communicate intraspecifically. Few studies, however, have examined how complex natural sounds are perceived in fishes. We investigated the representation of conspecific mating and agonistic calls in the auditory system of the Lusitanian toadfish Halobatrachus didactylus, and analysed auditory responses to heterospecific signals from ecologically relevant species: a sympatric vocal fish (meagre Argyrosomus regius) and a potential predator (dolphin Tursiops truncatus). Using auditory evoked potential (AEP) recordings, we showed that both sexes can resolve fine features of conspecific calls. The toadfish auditory system was most sensitive to frequencies well represented in the conspecific vocalizations (namely the mating boatwhistle), and revealed a fine representation of duration and pulsed structure of agonistic and mating calls. Stimuli and corresponding AEP amplitudes were highly correlated, indicating an accurate encoding of amplitude modulation. Moreover, Lusitanian toadfish were able to detect T. truncatus foraging sounds and A. regius calls, although at higher amplitudes. We provide strong evidence that the auditory system of a vocal fish, lacking accessory hearing structures, is capable of resolving fine features of complex vocalizations that are probably important for intraspecific communication and other relevant stimuli from the auditory scene.

Auditory saccular sensitivity of the vocal Lusitanian toadfish: low frequency tuning allows acoustic communication throughout the year.

A novel form of auditory plasticity for enhanced detection of social signals was described in a teleost fish, Porichthys notatus (Batrachoididae, Porichthyinae). The seasonal onset of male calling coincides with inshore migration from deep waters by both sexes and increased female sensitivity to dominant frequencies of male calls. The closely related Lusitanian toadfish, Halobatrachus didactylus, (Batrachoididae, Halophryninae) also breeds seasonally and relies on acoustic communication to find mates but, instead, both sexes stay in estuaries and show vocal activity throughout the year. We investigated whether the sensitivity of the inner ear saccule of H. didactylus is seasonally plastic and sexually dimorphic. We recorded evoked potentials from populations of saccular hair cells from non-reproductive and reproductive males and females in response to 15-945 Hz tones. Saccular hair cells were most sensitive at 15-205 Hz (thresholds between 111 and 118 dB re. 1 µPa). Both sexes showed identical hearing sensitivity and no differences were found across seasons. The saccule was well suited to detect conspecific vocalizations and low frequencies that overlapped with lateral line sensitivity. We showed that the saccule in H. didactylus has major importance in acoustic communication throughout the year and that significant sensory differences may exist between the two batrachoidid subfamilies.

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.