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.

Cortisol, cortisone and DHEAS in epidermis and scales of fish Aphanius fasciatus: HPLC-MS/MS measurement of stress indicators as proxies for natural and human-induced factors.

Fish health can be affected by a multitude of stressors. Acute and chronic stress assessment via specific hormones monitoring has become a trending research topic. Common investigated matrices are blood and plasma, but recently less invasive substrates have been identified. As chemical composition of skin mucus/epidermis has been demonstrated to link with acute stress, and of scales with chronic stress in fish, the aim of the study was firstly to improve the determination of three stress hormones, namely cortisol (COL), cortisone (CON), and dehydroepiandrosterone-3-sulfate (DHEAS), in skin mucus/epidermis and scales of Aphanius fasciatus. Secondly, an evaluation of the impact of different environments on hormones concentrations was carried out. A liquid chromatography coupled to tandem mass spectrometry method (HPLC-MS/MS) and a preanalytical procedure were validated to determine COL, CON and DHEAS. This methodology was applied to compare a pull of field-collected fish with a pull of fish housed in the laboratory for one year. Our results highlighted a significant presence of cortisol and cortisone in epidermis of the latter pull (averagely 0.10 and 0.14 ng mg-1, respectively), while in the first pull both hormones were much less concentrated (averagely 0.006 and 0.008 ng mg-1, respectively). Scales of both pulls showed presence of hormones, with a higher concentration for fish housed in the laboratory, although a relevant difference in concentration was found only for cortisone. DHEAS was always below the limit of detection.

Experimental investigation of multimodal noise generation by ducted low Mach number flows through orifice plates.

Noise generation by low Mach number air flows through circular orifices in rectangular ducts is investigated. In particular, the influence of the number and position of the orifices maintaining a constant flow area is addressed. A review of the available theories suggests a certain importance of such parameters in the excitation of higher-order acoustic duct modes. A qualitative coefficient is proposed for a first characterization of the ability to enhance or lessen a given higher-order acoustic mode by the plate geometry. An experimental campaign is performed to measure the total emitted acoustic power by different plate geometries as well as its modal composition. It is found that the orifices' numbers and positions greatly influence the acoustic emissions while the flow pressure drop caused by the obstacles is similar. The proposed qualitative coefficient shows good agreement with the experimental results. A particle image velocimetry measurement campaign is performed to visualize the near-field average flow behavior upstream and downstream of the orifice plates. An increase in the turbulent velocity fluctuations in the vicinity of the orifices is observed on both sides, further validating previous studies on the subject.

Boat-induced pressure does not influence breeding site selection of a vulnerable fish species in a highly anthropized coastal area.

The brown meagre (Sciaena umbra) is a vulnerable vocal fish species that may be affected by boat noise. The breeding site distribution along the anthropized Venice sea inlets was investigated, by using the species' chorusing activity as a proxy of spawning. Passive acoustic campaigns were repeated at 40 listening points distributed within the three inlets during three-time windows in both summer 2019 and 2020. The role of temporal, morphological, and hydrodynamic variables explaining the observed distribution patterns was evaluated using a GLM approach, considering also human-induced pressures among the candidate predictors. The GLM analysis indicates a higher probability of recording S. umbra chorus after sunset in deeper areas of the inlets, characterized by low water current, while the underwater noise overlapping the species' hearing range and boat abundance did not play any role. This suggests that the species' breeding site choice in the inlets was not influences by boat-induced pressure.

Correlation between acoustic divergence and phylogenetic distance in soniferous European gobiids (Gobiidae; Gobius lineage).

In fish, species identity can be encoded by sounds, which have been thoroughly investigated in European gobiids (Gobiidae, Gobius lineage). Recent evolutionary studies suggest that deterministic and/or stochastic forces could generate acoustic differences among related animal species, though this has not been investigated in any teleost group to date. In the present comparative study, we analysed the sounds from nine soniferous gobiids and quantitatively assessed their acoustic variability. Our interspecific acoustic study, incorporating for the first time the representative acoustic signals from the majority of soniferous gobiids, suggested that their sounds are truly species-specific (92% of sounds correctly classified into exact species) and each taxon possesses a unique set of spectro-temporal variables. In addition, we reconstructed phylogenetic relationships from a concatenated molecular dataset consisting of multiple molecular markers to track the evolution of acoustic signals in soniferous gobiids. The results of this study indicated that the genus Padogobius is polyphyletic, since P. nigricans was nested within the Ponto-Caspian clade, while the congeneric P. bonelli turned out to be a sister taxon to the remaining investigated soniferous species. Lastly, by extracting the acoustic and genetic distance matrices, sound variability and genetic distance were correlated for the first time to assess whether sound evolution follows a similar phylogenetic pattern. The positive correlation between the sound variability and genetic distance obtained here emphasizes that certain acoustic features from representative sounds could carry the phylogenetic signal in soniferous gobiids. Our study was the first attempt to evaluate the mutual relationship between acoustic variation and genetic divergence in any teleost fish.

Habitat constraints on carotenoid-based coloration in a small euryhaline teleost.

Display of bright and striking color patterns is a widespread way of communication in many animal species. Carotenoid-based coloration accounts for most of the bright yellow, orange, and red displays in invertebrates, fish, amphibians, reptiles, and birds, being widely considered a signal of individual health. This type of coloration is under the influence of several factors, such as sexual selection, predator pressure, pigment availability, and light transmission. Fish offer numerous examples of visual communication by means of color patterns. We used a small cyprinodontid fish, Aphanius fasciatus (Valenciennes, 1821), as a model species to assess habitat constraints on the color display in male caudal fin. Populations from natural and open/closed artificial habitats were tested for differences in the pigmentation of caudal fins. The most important factors explaining the intensity of coloration were the habitat type and the chlorophyll concentration in the sediment, followed by water turbidity; yellow fins were observed in natural habitats with low chlorophyll concentration and high water turbidity, while orange fins occurred in artificial habitats with high chlorophyll concentration and low turbidity. Furthermore, A. fasciatus in artificial habitats showed a higher somatic and a lower reproductive allotment with respect to natural habitats, according to the existing literature on the species. Furthermore, in closed artificial habitats, where the most intense reddish coloration of caudal fins was observed, a trade-off between somatic growth and the coloration intensity of a carotenoid-based sexual ornament has been observed; in these populations, intensity of caudal fin coloration was negatively related to the somatic allotment. Results of this study suggested how both the pigmentation of male's caudal fin and the life history strategies of the species are constrained by habitat characteristics.

Convergent Aspects of Acoustic Communication in Darters, Sculpins, and Gobies.

Darters (Perciformes, Percidae), sculpins (Perciformes, Cottidae), and gobioids (Gobiiformes, Gobioidei) exhibit convergent life history traits, including a benthic lifestyle and a cavity nesting spawning mode. Soniferous species within these taxa produce pulsed and/or tonal sounds with peak frequencies below 200 Hz (with some exceptions), primarily in agonistic and/or reproductive contexts. The reduced or absent swim bladders found in these taxa limit or prevent both hearing enhancement via pressure sensitivity and acoustic amplification of the contracting sonic muscles, which are associated with the skull and pectoral girdle. While such anatomies constrain communication to low frequency channels, optimization of the S/N (signal-to-noise) ratio in low frequency channels is evident for some gobies, as measured by habitat soundscape frequency windows, nest cavity sound amplification, and audiograms. Similar S/N considerations are applicable to many darter and sculpin systems. This chapter reviews the currently documented diversity of sound production in darters, sculpins, and gobioids within a phylogenetic context, examines the efficacy of signal transmission from senders to receivers (sound production mechanisms, audiograms, and masking challenges), and evaluates the potential functional significance of sound attributes in relation to territorial and reproductive behaviours.

Is the Venice Lagoon Noisy? First Passive Listening Monitoring of the Venice Lagoon: Possible Effects on the Typical Fish Community.

Three passive listening surveys have been carried out in two of the three Venice lagoon tide inlets and inside the Venice island. The spectral content and the intensity level of the underwater noise as well as the presence or absence of Sciaena umbra and the distribution of its different sound patterns have been investigated in all the recording sites. The passive listening proved to be successful in detecting S. umbra drumming sounds in both Venice lagoon tide inlets. Our results indicate that the spectral content and the level of underwater noise pollution in the Venice lagoon could affect fish acoustic communication.

Quantitative measurement of blood velocity in zebrafish with optical vector field tomography.

Microscopy techniques can readily visualize the finest details of embryo vasculature, but still lack to provide a complete three-dimensional representation of blood flow parameters. We present an in-vivo 3D imaging technique, able to reconstruct the blood cell velocity vector over a large volume of zebrafish embryos. This low cost and relatively simple technique is exploited to quantitatively assess blood velocity in the zebrafish tail at different stages of development.

Sound production mechanism in Gobius paganellus (Gobiidae).

Gobiidae, the largest fish family (>1500 species), has species from at least 10 genera that produce sounds for communication. Studies focused on goby sound production mechanisms have suggested that sounds are produced by the forcible ejection of water through small apertures in the opercles (hydrodynamic mechanism). The present study was a multidisciplinary investigation (morphology, muscle histology, high-speed video, sound analysis and electromyography) of the sound emission mechanism in Gobius paganellus, which produces both pulsed and tonal calls. Two populations were used, from Brittany and Venice. In the French population, sounds were accompanied by a suite of coordinated movements of the buccal, branchial and opercular regions. This was not the case in the Venetian population, and thus the direct role of head movements in sound production was rejected. The hydrodynamic mechanism hypothesis was also rejected in G. paganellus on the basis of sound oscillogram shape and because sounds are still produced after the opercles and hyohyoid muscles are cut. The use of both electromyography and electron microscopy showed that the levator pectoralis muscle, which originates on the skull and inserts on the dorsal tip of the cleithrum, is involved in sound production. We propose that the contraction of this muscle and associated vibration of the large radials is used to make sounds. In addition, we propose that different sound types (pulsed sounds and tonal calls) could occur because of differences in fish size.

Acoustic communication at the water's edge: evolutionary insights from a mudskipper.

Coupled behavioural observations and acoustical recordings of aggressive dyadic contests showed that the mudskipper Periophthalmodon septemradiatus communicates acoustically while out of water. An analysis of intraspecific variability showed that specific acoustic components may act as tags for individual recognition, further supporting the sounds' communicative value. A correlative analysis amongst acoustical properties and video-acoustical recordings in slow-motion supported first hypotheses on the emission mechanism. Acoustic transmission through the wet exposed substrate was also discussed. These observations were used to support an "exaptation hypothesis", i.e. the maintenance of key adaptations during the first stages of water-to-land vertebrate eco-evolutionary transitions (based on eco-evolutionary and palaeontological considerations), through a comparative bioacoustic analysis of aquatic and semiterrestrial gobiid taxa. In fact, a remarkable similarity was found between mudskipper vocalisations and those emitted by gobioids and other soniferous benthonic fishes.

A cytogenetical study on Economidichthys pygmaeus Holly, 1929 (Pisces, Gobiidae), an endemic freshwater goby from Western Greece.

A cytogenetic study was carried out on the chromosomes and the nuclear DNA content of the freshwater goby Economidichthys pygmaeus (Pisces, Gobiidae). The species is characterized by a 2n=46 karyotype consisting of 12 submetacentric and 11 subtelocentric chromosome pairs (NF=70). Major (45S) rDNA genes are terminal-centromeric located on the short arm of a single medium-small sized submetacentric pairas assessed by in situ hybridization, CMA3 staining, and Ag-NOR banding. The haploid (C-value) nuclear DNA content is 0.93±0.003 picograms. The cytogenetical data of Economidichthys pygmaeus were compared with those ones already available for other related gobies.