Modulation of whistle production related to training sessions in bottlenose dolphins (Tursiops truncatus) under human care.

Bottlenose dolphins are highly social cetaceans with an extensive sound production including clicks, burst-pulsed sounds, and whistles. Some whistles, known as signature whistles, are individually specific. These acoustic signatures are commonly described as being emitted in contexts of stress during forced isolation and as group cohesion calls. Interactions between humans and captive dolphins is largely based on positive reinforcement conditioning within several training/feeding sessions per day. Vocal behavior of dolphins during these interactions might vary. To investigate this, we recorded 10 bottlenose dolphins of Parc Asterix dolphinarium (France) before, during and after 10 training sessions for a total duration of 7 hr and 32 min. We detected 3,272 whistles with 2,884 presenting a quality good enough to be categorized. We created a catalog of whistle types by visual categorization verified by five naive judges (Fleiss' Kappa Test). We then applied the SIGID method to identify the signatures whistles present in our recordings. We found 279 whistles belonging to one of the four identified signature whistle types. The remaining 2,605 were classified as non-signature whistles. The non-signature whistles emission rate was higher during and after the training sessions than before. Emission rate of three signature whistles types significantly increased afterwards as compared to before the training sessions. We suggest that dolphins use their signature whistles when they return to their intraspecific social interactions succeeding scheduled and human-organized training sessions. More observations are needed to make conclusions about the function of signature whistles in relation to training sessions. Zoo Biol. 35:495-504, 2016. © 2016 Wiley Periodicals, Inc.

Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry.

Infection by dengue flavivirus is transmitted by mosquitoes and affects tens to hundreds of millions people around the world each year. Four serotypes have been described, all of which cause similar disease. Currently, there no approved vaccines or specific therapeutics for dengue, although several vaccine prototypes are in different stages of clinical development. Among them, a chimeric vaccine, built from the replication machinery of the yellow fever 17D virus, has shown promising results in phase III trials. Accurate quantitation of expressed viral particles in alive attenuated viral antigen vaccine is essential and determination of infectious titer is usually the method of choice. The current paper describes an alternative or orthogonal strategy, namely, a multiplexed and absolute assay of four proteins of the chimera yellow fever/dengue serotype 4 virus using targeted MS in SRM mode. Over 1 month, variability of the assay using a partially purified Vero cell extract was between 8 and 17%, and accuracy was between 80 and 120%. In addition, the assay was linear between 6.25 and 200 nmol/L and could therefore be used in the near future to quantify dengue virus type 4 during production and purification from Vero cells.

Treatment of influenza virus with beta-propiolactone alters viral membrane fusion.

Beta-propiolactone (BPL) is commonly used as an inactivating reagent to produce viral vaccines. Although BPL has been described to chemically modify nucleic acids, its effect on viral proteins, potentially affecting viral infectivity, remains poorly studied. Here, a H3N2 strain of influenza virus was submitted to treatment with various BPL concentrations (2-1000µM). Cell infectivity was progressively reduced and entirely abolished at 1mM BPL. Virus fusion with endosome being a critical step in virus infection, we analyzed its ability to fuse with lipid membrane after BPL treatment. By monitoring calcein leakage from liposomes fusing with the virus, we measured a decrease of membrane fusion in a BPL dose-dependent manner that correlates with the loss of infectivity. These data were complemented with cryo transmission electron microscopy (cryoTEM) and cryo electron tomography (cryoET) studies of native and modified viruses. In addition, a decrease of leakage irrespective of BPL concentration was measured suggesting that the insertion of HA2 fusion peptide into the target membrane was inhibited even at low BPL concentrations. Interestingly, mass spectrometry revealed that HA2 and M1 matrix proteins had been modified. Furthermore, fusion activity was partially restored by the protonophore monensin as confirmed by cryoTEM and cryoET. Moreover, exposure to amantadine, an inhibitor of M2 channel, did not alter membrane fusion activity of 1mM BPL treated virus. Taken together these results show that BPL treatment inhibits membrane fusion, likely by altering function of proteins involved in the fusion process, shedding new light on the effect of BPL on influenza virus.

An investigation of prior knowledge in Automatic Music Transcription systems.

Automatic transcription of music is a long-studied research field with many operational systems available commercially. In this paper, a generic transcription system able to host various prior knowledge parameters has been developed, followed by an in-depth investigation of their impact on music transcription. Explicit links between musical knowledge and algorithmic formalism have been made. Musical knowledge covers classes of timbre, musicology, and playing style of an instrument repertoire. An evaluation sound corpus gathering musical pieces played by human performers from three different instrument repertoires, namely, classical piano, steel-string acoustic guitar, and the marovany zither from Madagascar, has been developed. The different components of musical knowledge have been successively incorporated in a complete transcription system, consisting mainly of a Probabilistic Latent Component Analysis algorithm post-processed with a Hidden Markov Model, and their impact on transcription results have been comparatively evaluated.

Mimicking influenza virus fusion using supported lipid bilayers.

Influenza virus infection is a serious public health problem in the world, and understanding the molecular mechanisms involved in viral replication is crucial. In this paper, we used a minimalist approach based on a lipid bilayer supported on mica, which we imaged by atomic force microscopy (AFM) in a physiological buffer, to analyze the different steps of influenza fusion, from the interaction of intact viruses with the supported bilayer to their complete fusion. Our results show that sialic acid recognition and priming upon acidification are sufficient for a complete fusion with the host cell membrane. After fusion, a flat and continuous membrane was observed. Because of the fragility of the viral membrane that was removed by the tip, most probably due to the disorganization of the matrix layer at acidic pH, fine structural details of ribonucleoproteins (RNP) were obtained. In addition, AFM topography of intact virus in interaction with the supported lipid bilayer confirms that hemeagglutinin and neuraminidase can form isolated clusters within the viral membrane.

Vocal complexity in a socially complex corvid: gradation, diversity and lack of common call repertoire in male rooks.

Vocal communication is widespread in animals, with vocal repertoires of varying complexity. The social complexity hypothesis predicts that species may need high vocal complexity to deal with complex social organization (e.g. have a variety of different interindividual relations). We quantified the vocal complexity of two geographically distant captive colonies of rooks, a corvid species with complex social organization and cognitive performances, but understudied vocal abilities. We quantified the diversity and gradation of their repertoire, as well as the inter-individual similarity at the vocal unit level. We found that males produced call units with lower diversity and gradation than females, while song units did not differ between sexes. Surprisingly, while females produced highly similar call repertoires, even between colonies, each individual male produced almost completely different call repertoires from any other individual. These findings question the way male rooks communicate with their social partners. We suggest that each male may actively seek to remain vocally distinct, which could be an asset in their frequently changing social environment. We conclude that inter-individual similarity, an understudied aspect of vocal repertoires, should also be considered as a measure of vocal complexity.

Understanding the intentional acoustic behavior of humpback whales: a production-based approach.

Following a production-based approach, this paper deals with the acoustic behavior of humpback whales. This approach investigates various physical factors, which are either internal (e.g., physiological mechanisms) or external (e.g., environmental constraints) to the respiratory tractus of the whale, for their implications in sound production. This paper aims to describe a functional scenario of this tractus for the generation of vocal sounds. To do so, a division of this tractus into three different configurations is proposed, based on the air recirculation process which determines air sources and laryngeal valves. Then, assuming a vocal function (in sound generation or modification) for several specific anatomical components, an acoustic characterization of each of these configurations is proposed to link different spectral features, namely, fundamental frequencies and formant structures, to specific vocal production mechanisms. A discussion around the question of whether the whale is able to fully exploit the acoustic potential of its respiratory tractus is eventually provided.

Characterizing the suckling behavior by video and 3D-accelerometry in humpback whale calves on a breeding ground.

Getting maternal milk through nursing is vital for all newborn mammals. Despite its importance, nursing has been poorly documented in humpback whales (Megaptera novaeangliae). Nursing is difficult to observe underwater without disturbing the whales and is usually impossible to observe from a ship. We attempted to observe nursing from the calf's perspective by placing CATS cam tags on three humpback whale calves in the Sainte Marie channel, Madagascar, Indian Ocean, during the breeding seasons. CATS cam tags are animal-borne multi-sensor tags equipped with a video camera, a hydrophone, and several auxiliary sensors (including a 3-axis accelerometer, a 3-axis magnetometer, and a depth sensor). The use of multi-sensor tags minimized potential disturbance from human presence. A total of 10.52 h of video recordings were collected with the corresponding auxiliary data. Video recordings were manually analyzed and correlated with the auxiliary data, allowing us to extract different kinematic features including the depth rate, speed, Fluke Stroke Rate (FSR), Overall Body Dynamic Acceleration (ODBA), pitch, roll, and roll rate. We found that suckling events lasted 18.8 ± 8.8 s on average (N = 34) and were performed mostly during dives. Suckling events represented 1.7% of the total observation time. During suckling, the calves were visually estimated to be at a 30-45° pitch angle relative to the midline of their mother's body and were always observed rolling either to the right or to the left. In our auxiliary dataset, we confirmed that suckling behavior was primarily characterized by a high average absolute roll and additionally we also found that it was likely characterized by a high average FSR and a low average speed. Kinematic features were used for supervised machine learning in order to subsequently detect suckling behavior automatically. Our study is a proof of method on which future investigations can build upon. It opens new opportunities for further investigation of suckling behavior in humpback whales and the baleen whale species.

Social calls in humpback whale mother-calf groups off Sainte Marie breeding ground (Madagascar, Indian Ocean).

Humpback whales (Megaptera novaeangliae) use vocalizations during diverse social interactions or activities such as foraging or mating. Unlike songs produced only by males, social calls are produced by all types of individuals (adult males and females, juveniles and calves). Several studies have described social calls in the humpback whale's breeding and the feeding grounds and from different geographic areas. We aimed to investigate for the first time the vocal repertoire of humpback whale mother-calf groups during the breeding season off Sainte Marie island, Madagascar, South Western Indian Ocean using data collected in 2013, 2014, 2016, and 2017. We recorded social calls using Acousonde tags deployed on the mother or the calf in mother-calf groups. A total of 21 deployments were analyzed. We visually and aurally identified 30 social call types and classified them into five categories: low, medium, high-frequency sounds, amplitude-modulated sounds, and pulsed sounds. The aural-visual classifications have been validated using random forest (RF) analyses. Low-frequency sounds constituted 46% of all social calls, mid-frequency 35%, and high frequency 10%. Amplitude-modulated sounds constituted 8% of all vocalizations, and pulsed sounds constituted 1%. While some social call types seemed specific to our study area, others presented similarities with social calls described in other geographic areas, on breeding and foraging grounds, and during migrating routes. Among the call types described in this study, nine call types were also found in humpback whale songs recorded in the same region. The 30 call types highlight the diversity of the social calls recorded in mother-calf groups and thus the importance of acoustic interactions in the relationships between the mother and her calf and between the mother-calf pair and escorts.

Three dimensional morphology of rabies virus studied by cryo-electron tomography.

The rabies virus (RABV) continues to be a worldwide health problem. RABV contains a single-stranded RNA genome that associates with the nucleoprotein N. The resulting ribonucleoprotein complex is surrounded by matrix protein M, lipid bilayer and glycoprotein G. RABV was reported to organize in bullet-like virions, but the role of each viral component in adopting this morphology is unclear. We present here a cryo-electron tomography study of RABV showing additional morphologies consisting in bullet-like virions containing a tubular, lipidic appendage having G-protein at its apex. In addition, there was evidence for an important fraction of pleomorphic particles. These pleomorphic forms differed in the amount of membrane-associated M-, M/N-protein providing interesting insight into its role in viral morphogenesis. In the absence of membrane-associated M-, M/N-protein viral morphology was almost spherical. Other images, showing straight membrane portions, correlate with the M-protein recruitment at the membrane independently of the presence of the G-protein. The viral membrane was found to contain a negative net charge indicating that M-, M/N-protein-membrane charge attraction drives this interaction.

Acoustic behaviour of bottlenose dolphins under human care while performing synchronous aerial jumps.

Synchronous behaviours occur when two or more animals display the same behaviour at the same time. However, the mechanisms underlying this synchrony are not well understood. In this study, we carried out an experiment to determine whether or not Bottlenose dolphins use acoustic cues when performing a known synchronised exercise. For this, we recorded three dolphins while they performed requested aerial jumps both individually or synchronously in pairs, with a hydrophone array and a 360° underwater video camera allowing the identification of the subject emitting vocalisations. Results indicated that in pairs, dolphins synchronised their jumps 100% of the time. Whether they jumped alone or in pairs, they produced click trains before and after 92% of jumps. No whistles or burst-pulsed sounds were emitted by the animals during the exercise. The acoustic localisation process allowed the successful identification of the vocalising subject in 19.8% of all cases (N = 141). Our study showed that in all (n = 28) but one successful localisations, the click trains were produced by the same individual. It is worth noting that this individual was the oldest female of the group. This paper provides evidence suggesting that during synchronous behaviours, dolphins use acoustic cues, and more particularly click trains, to coordinate their movements; possibly by eavesdropping on the clicks or echoes produced by one individual leading the navigation.

Bidirectional Interactions With Humpback Whale Singer Using Concrete Sound Elements.

We describe an art-science project called "Feral Interactions-The Answer of the Humpback Whale" inspired by humpback whale songs and interactions between individuals based on mutual influences, learning process, or ranking in the dominance hierarchy. The aim was to build new sounds that can be used to initiate acoustic interactions with these whales, not in a one-way direction, as playbacks do, but in real interspecies exchanges. Thus, we investigated how the humpback whales generate sounds in order to better understand their abilities and limits. By carefully listening to their emitted vocalizations, we also describe their acoustic features and temporal structure, in a scientific way and also with a musical approach as it is done with musique concrète, in order to specify the types and the morphologies of whale sounds. The idea is to highlight the most precise information to generate our own sounds that will be suggested to the whales. Based on the approach developed in musique concrète, similarities with the sounds produced by bassoon were identified and then were processed to become "concrete sound elements." This analysis also brought us to design a new music interface that allows us to create adapted musical phrases in real-time. With this approach, interactions will be possible in both directions, from and to whales.

Kin relationships in cultural species of the marine realm: case study of a matrilineal social group of sperm whales off Mauritius island, Indian Ocean.

Understanding the organization and dynamics of social groups of marine mammals through the study of kin relationships is particularly challenging. Here, we studied a stable social group of sperm whales off Mauritius, using underwater observations, individual-specific identification, non-invasive sampling and genetic analyses based on mitochondrial sequencing and microsatellite profiling. Twenty-four sperm whales were sampled between 2017 and 2019. All individuals except one adult female shared the same mitochondrial DNA (mtDNA) haplotype-one that is rare in the western Indian Ocean-thus confirming with near certainty the matrilineality of the group. All probable first- and second-degree kin relationships were depicted in the sperm whale social group: 13 first-degree and 27 second-degree relationships were identified. Notably, we highlight the likely case of an unrelated female having been integrated into a social unit, in that she presented a distinct mtDNA haplotype and no close relationships with any members of the group. Investigating the possible matrilineality of sperm whale cultural units (i.e. vocal clans) is the next step in our research programme to elucidate and better apprehend the complex organization of sperm whale social groups.

High sensitivity of Gammarus sp. juveniles to deltamethrin: outcomes for risk assessment.

Amphipods from the genus Gammarus are among the most frequently used organisms in ecotoxicological testing and in situ bioindication. Because of their importance in risk assessment, it is necessary to understand the potential roles of life stage, population source and inter-specific differences on the sensitivity of these organisms to contaminants. In the present study, the acute toxicity of deltamethrin, a commonly used pyrethroid insecticide, has been tested for Gammarus fossarum and Gammarus pulex (Crustacea, Amphipoda) to document the inter- and intra-specific variability at different developmental stages. Adult G. fossarum were about two-fold more sensitive to deltamethrin than adult G. pulex, 96-h LC50 being 33.2 and 68.0 ng L(-1), respectively. However, in the same species, significant differences of sensitivity were observed between individuals from different locations. Furthermore, G. fossarum from certain localities were less sensitive to deltamethrin than certain G. pulex. In addition, juveniles of both species were about 14- to 22-fold more sensitive to deltamethrin than adults: 48-h LC50 in G. fossarum and G. pulex juveniles were 4.0 and 5.7 ng L(-1). Therefore, lethal effects of deltamethrin on Gammarus sp. populations likely depend more on juvenile response rather than on adult response. Since juveniles were also the most abundant population component, the present results show that risk assessment should consider at this developmental stage.

Source level estimation of two blue whale subspecies in southwestern Indian Ocean.

Blue whales produce intense, stereotypic low frequency calls that are particularly well suited for transmission over long distances. Because these calls vary geographically, they can be used to gain insight into subspecies distribution. In the Southwestern Indian Ocean, acoustic data from a triad of calibrated hydrophones maintained by the International Monitoring System provided data on blue whale calls from two subspecies: Antarctic and pygmy blue whales. Using time difference of arrival and least-squares hyperbolic methods, the range and location of calling whales were determined. By using received level of calls and propagation modeling, call source levels of both subspecies were estimated. The average call source level was estimated to 179+/-5 dB re 1 microPa(rms) at 1 m over the 17-30 Hz band for Antarctic blue whale and 174+/-1 dB re 1 microPa(rms) at 1 m over the 17-50 Hz band for pygmy blue whale. According to previous estimates, slight variations in the source level could be due to inter-individual differences, inter-subspecies variations and the calculation method. These are the first reported source level estimations for blue whales in the Indian Ocean. Such data are critical to estimate detection ranges of calling blue whales.

Contexts of emission of non-signature whistles in bottlenose dolphins (Tursiops truncatus) under human care.

Bottlenose dolphins are social cetaceans that strongly rely on acoustic communication and signaling. The diversity of sounds emitted by the species has been structurally classified into whistles, clicks and burst-pulsed sounds. Although click sounds and individually-specific signature whistles have been largely studied, not much is known about non-signature whistles. Most studies that link behavior and whistle production conduct aerial behavioral observations and link the production of whistles to the general category of social interactions. The aim of this study was to determine if there was a correlation between the non-signature whistle production and the underwater behaviors of a group of bottlenose dolphins (Tursiops truncatus) under human care, during their free time in the absence of trainers. To do this we made audio-video recordings 15 min before and after 10 training sessions of eight dolphins in Boudewijn Seapark (Belgium). For the behavioral analysis we conducted focal follows on each individual based on six behavioral categories. For the acoustical analysis, carried out at the group level, we used the SIGID method to identify non-signature whistles (N = 661) and we classified them in six categories according to their frequency modulation. The occurrences of the six categories of whistles were highly collinear. Most importantly, non-signature whistle production was positively correlated with the time individuals spent slow swimming alone, and was negatively correlated with the time spent in affiliative body contact. This is the first analysis that links the production of non-signature whistles with particular underwater behaviors in this species.

In Situ Reproductive Bioassay with Caged Gammarus fossarum (Crustacea): Part 1-Gauging the Confounding Influence of Temperature and Water Hardness.

Monitoring the adverse effects of environmental contaminants on the reproduction of invertebrate species in the field remains a challenge in aquatic ecotoxicology. To meet the need for reliable tools for in situ toxicity assessment, we present the first part of a methodological study of the in situ implementation of a reproductive bioassay in Gammarus previously developed for screening the toxicity of chemical compounds during laboratory exposure. To ensure the correct interpretation of the modulation of reproductive markers (molting, fecundity, follicle growth, and embryonic development) in uncontrolled environmental conditions, we experimentally assessed and statistically modeled the variability in the female reproductive cycle during laboratory exposure under several temperature and water hardness conditions. Whereas water hardness did not influence the reproductive cycle, the significant accelerating effect of temperature on the dynamics of molting and marsupial development was finely modeled, by detailing the influence of temperature on the probability of transition between all molt and embryonic stages along the female cycle. In addition, no effect of temperature or water hardness was detected on the number of oocytes and embryos carried by females. Furthermore, the finding that the relative durations of the first 4 molt and embryonic stages are constant whatever the temperature makes it possible to predict the molting dynamics in fluctuating temperature conditions. Because this could allow us to take into account the confounding influence of temperature on the measurement of reproductive markers, the implications of these findings for an optimal in situ implementation of the reproductive bioassay with G. fossarum are discussed. The relevance of this modeling approach during in situ implementation is tested in a companion study. Environ Toxicol Chem 2020;39:667-677. © 2019 SETAC.

Mixture toxicity assessment of wood preservative pesticides in the freshwater amphipod Gammarus pulex (L.).

All over the world, insecticides and fungicides are used to protect wood against pathogens. To document the environmental toxicity of wood preservative mixtures, freshwater amphipods Gammarus pulex (L.) were submitted to organic pesticides given independently or in mixtures. When given independently at environmentally realistic concentrations, propiconazole and tebuconazole (triazoles fungicides) were not toxic for G. pulex, 3-iodo-2-propinyl butyl carbamate (IPBC, fungicide) was moderately toxic, and cypermethrin (pyrethroid insecticide) was extremely toxic. 96-h LC50 were, respectively, 4703, 1643, 604, and 0.09 microg L(-1). When amphipods were submitted to a mixture mimicking the composition of a commercial solution (18.2% of cypermethrin, 45.8% propiconazole, 17.2% tebuconazole, 18.8% IPBC), the overall toxicity was equal to that of the most toxic component, namely cypermethrin. But, when organisms were submitted to the real commercial mixture containing pesticides, solvents and additives, the toxic effects were markedly higher. Moreover, a third mixture with only 0.002% cypermethrin showed lethality 2.5-18-fold higher than those predicted by the commonly used models. The present results show that toxicity of wood preservative mixtures cannot be assessed starting only from the toxicities of each single component. Furthermore, the present data strongly suggest that the environmental impacts of wood preservative mixtures might be frequently underestimated.

Anatomy and Functional Morphology of the Mysticete Rorqual Whale Larynx: Phonation Positions of the U-Fold.

Many Mysticetes (baleen whales) are acoustically active marine mammals. This is epitomized by rorquals, and specifically male humpback whales (Megaptera novaeangliae) whose complex songs comprise a wide range of vocalizations. The sound production mechanism of odontocetes (toothed whales, including dolphins and porpoises) is well described, in contrast to that of mysticetes whose vocalization mechanism remains a subject of active scientific investigation. Anatomical observations and acoustic signal processing have led to divergent hypotheses under the framework of a production-based approach. We attempt to unify these hypotheses by broadening existing data with our new anatomical investigation, interpreted in light of known acoustical properties of mysticete vocalizations. We examined 15 specimens of four rorqual species: sei whale (Baleanoptera borealis), fin whale (Baleanoptera physalus), minke whale (Baleanoptera acutorostrata), and humpback whale (Megaptera novaeangliae). Based on these data and on previous literature, we propose a description of three functional positions (rest, breathing, and recirculation), unidirectional egressive airflow for sound production (from lungs to laryngeal sac), and new nomenclature for different parts of the U-fold (distal section, midsection, and corniculate flaps). Each of these sections has specific morphological and acoustical properties that support the concept of "mode variation" in baleen whale vocalizations. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 302:703-717, 2019. © 2018 Wiley Periodicals, Inc.

The removal of Basic Blue 3 from aqueous solutions by chitosan-based adsorbent: batch studies.

Chitosan-based adsorbent (CHITOD material) is used for the removal of Basic Blue 3 (BB 3) from aqueous solutions. The adsorption of BB 3 on CHITOD material was studied as a function of time, sorbent mass and concentration. The influence of these parameters on the adsorption capacity was evaluated using the batch method. Results of adsorption experiments and kinetic data showed that (i) the CHITOD adsorbent exhibited high sorption capacities toward BB 3; (ii) the Langmuir equation represented the best fit of experimental data; (iii) the dye sorption on material was exothermic and spontaneous in nature; (iv) the kinetic measurements showed that the process was rapid; (v) the adsorption kinetics followed a pseudo-second order model; and (vi) the sorption was dependent on the presence of sulfonate groups. Non-linear method was also found to be more appropriate method for estimating the isotherm and kinetic parameters.