Morpho-functionality of the toothed whale external ear canal

Tesi en modalitat de cotutela: Universitat Politècnica de Catalunya i Universita degli studi di PadovaWhile marine, anthropogenic noise pollution is a scientific and societal matter of concern, there is limited knowledge on how sea animals, particularly cetaceans, perceive their environment through sounds. Toothed whales, like all cetaceans, show a series of astonishing morphological and physiological evolutionary adaptations, of which one of the most striking can be found in the complex configuration of the hearing apparatus. The external ear canal, although no longer considered a direct actor in the sound reception process, also shows surprising adaptation with many active structures and a complex peripheral innervation, although basic knowledge on its morphology is inconclusive at the moment. This study aimed at providing fundamental knowledge on the morphology of the external ear canal in various toothed whales, associated to its sensory capabilities with in-depth morphological descriptions of its shape and course, lumen and content, epithelium, glands, lymphoid tissue, vascularization, innervation, muscular tissue, cartilage, fat and connective tissue. Furthermore, specific attention was given to the identification and morphological characterization of the sensory formations associated with the external ear canal, and the comparison with the terrestrial cetartiodactyl external ear canal, to gain perspective of a comprehensive understanding of the cetacean sensory abilities. Post-mortem samples were gathered during necropsies of wild toothed whales, in an international collaborative effort. The tissues were inspected macroscopically and subjected to microscopic studies including immunohistochemical analyses using antibodies specific for nervous tissue, various histochemical techniques, ultrastructural investigation using transmission electron microscopy, and 3D reconstruction from histological slides of the ear canal and associated tissues. The winding structure of the external ear canal revealed a complex organ that comprises a physiological function reflected in its delicate anatomical structures, all of which are discussed in detail. Remarkably, the innervation showed an extensive intramural nervous plexus with the predominant presence of simple lamellar corpuscles, similar to Pacinian corpuscles although without an outer core or capsule. There were differences in conformation along the canal, from a network that fully encompasses the ear canal to a nervous tissue ridge that bulges into the lumen. The work elaborates on hypotheses related to the external ear canal’s function and the somatosensory system in toothed whales, taking into account the importance of the perspective of sensory modalities in the marine environment. The results indicate it plays an important physiological function, which impairment may have direct effects on their sensory capabilities and compromise essential physiological processes. It also puts into question to what extent there might be an adverse effect from various sources of anthropogenic noise, as it can cause physical changes in the sensory tissues in cetaceans and other marine fauna.Morfo-funcionalitat del canal auditiu extern en odontocets. Si bé la contaminació acústica marina antropogènica és un assumpte que preocupa la comunitat científica i la societat en general, el coneixement sobre com els animals marins, en particular els cetacis, perceben el seu entorn a través dels sons és limitat. Els odontocets, com tots els cetacis, presenten una sèrie de sorprenents adaptacions evolutives morfològiques i fisiològiques. Entre elles, una de les més notables es troba en la complexa configuració de l'aparell auditiu. El canal auditiu extern, tot i que no es considera que tingui un paper directe en el procés de recepció de el so, també mostra una sorprenent adaptació al medi marí, mostrant moltes estructures actives i una complexa innervació perifèrica. Malgrat això, els coneixements bàsics sobre la seva morfologia no són concloents de moment. Aquest estudi tenia com a objectiu la descripció fonamental de la morfologia del conducte auditiu extern en diversos odontocets, associant-la a les seves capacitats sensorials a través de descripcions morfològiques en profunditat de la seva forma i curs, lumen i contingut, epiteli, glàndules, teixit limfoide, vascularització, innervació, teixit muscular, cartílag, teixit adipós i connectiu. A més, es va parar especial atenció a la identificació i caracterització morfològica de les formacions sensorials associades al canal auditiu extern, i es va fer una comparació amb el conducte auditiu extern de cetartiodáctilos terrestres, per tal d'assolir una comprensió integral de les capacitats sensorials dels cetacis. Es van recol·lectar mostres post-mortem durant necròpsies d¿odontocets salvatges, en un esforç de col·laboració internacional. Els teixits es van inspeccionar macroscòpicament i es van sotmetre a estudis microscòpics, incloent anàlisis immunohistoquímics amb anticossos específics per teixit nerviós, diverses tècniques histoquímiques, investigació ultraestructural mitjançant microscòpia electrònica de transmissió i reconstrucció 3D a partir de talls histològics de canal auditiu i teixits associats. L'estructura sinuosa de canal auditiu extern va revelar un òrgan complex, la funció fisiològica del qual es reflecteix en les seves delicades estructures anatòmiques, que presenten una morfologia que es discuteix en detall. Sorprenentment, la innervació va mostrar un extens plexe nerviós intramural amb la presència predominant de corpuscles lamelares simples, similars als corpuscles de Pacini, encara que sense capa externa o càpsula. Es van observar diferències en la conformació dels corpuscles al llarg del canal auditiu, des d'una xarxa que abasta completament el conducte fins a una cresta de teixit nerviós que sobresurt en el lumen. El treball desenvolupa hipòtesis relacionades amb la funció del conducte auditiu extern i el sistema somatosensorial en odontocets, tenint en compte la importància de la perspectiva de les modalitats sensorials en el medi marí. Els resultats indiquen que el canal exerceix una funció fisiològica important, el deteriorament del qual pot tenir efectes directes sobre les seves capacitats sensorials i comprometre processos fisiològics essencials. També qüestiona fins a quin punt les diverses fonts de soroll antropogènic poden tenir un efecte advers, ja que poden provocar canvis físics en els teixits sensorials en cetacis i altra fauna marinaStudio morfo-funzionale del canale uditivo esterno negli odontoceti. Nonostante l'inquinamento acustico marino sia una questione che induce una crescente preoccupazione scientifica e sociale, la conoscenza su come gli animali marini, in particolare i cetacei, percepiscono il loro ambiente attraverso i suoni è ancora limitata. Gli odontoceti in particolare mostrano una serie di notevoli adattamenti evolutivi morfologici e fisiologici, di cui uno dei più eclatanti si trova nella complessa configurazione dell'apparato uditivo. Il condotto uditivo esterno, sebbene non sia più considerato una parte direttamente interessata nel processo di ricezione del suono, mostra un sorprendente adattamento con molte strutture attive e una complessa innervazione periferica. Tuttavia, le conoscenze basilari sulla sua morfologia e funzione sono incomplete al momento. Questo studio mira ad approfondire la morfologia del condotto uditivo esterno in vari odontoceti, con descrizioni morfologiche fornendo dettagli sulla sua forma e decorso, lume e contenuto, epitelio, ghiandole, tessuto linfoide, vascolarizzazione, innervazione, tessuto muscolare, cartilagine, tessuto adiposo e connettivo. Inoltre, una particolare attenzione è stata data all'identificazione e caratterizzazione morfologica delle formazioni sensoriali associate al condotto uditivo esterno, e al confronto con la medesima struttura in altri ceto-artiodattili terrestri, per ottenere una prospettiva di comprensione complessiva delle capacità sensoriali dei cetacei. I campioni sono stati raccolti durante necroscopie di odontoceti spiaggiati, in uno sforzo di collaborazione internazionale. I tessuti sono stati valutati macroscopicamente e quindi sottoposti a studi microscopici, comprendenti analisi immunoistochimiche utilizzando anticorpi specifici per il tessuto nervoso, insieme a varie tecniche istochimiche e indagini ultrastrutturali mediante microscopia elettronica a trasmissione. Ciò ha inoltre portato alla ricostruzione 3D partendo da vetrini istologici del condotto uditivo e dei tessuti associati. Il condotto uditivo esterno si è rivelato un organo complesso che comprende una funzione fisiologica riflessa nella sua delicata struttura anatomica. L'innervazione mostrava un esteso plesso nervoso intramurale con la presenza predominante di semplici corpuscoli lamellari, simili ai corpuscoli del Pacini, sebbene senza strato esterno o capsula. Lungo il decorso del condotto si sono notate alcune differenze nella sua architettura e distribuzione, da una rete che racchiude completamente il condotto uditivo a una papilla di tessuto nervoso che si gonfia nel lume. Grazie a questo studio si è potuto, al termine del lavoro, elaborare ipotesi relative alla funzione del condotto uditivo esterno e del sistema somatosensoriale nelle balene dentate, tenendo conto dell'importanza della prospettiva delle modalità sensoriali nell'ambiente marino. I risultati indicano che svolge una rilevante funzione fisiologica, la cui compromissione può avere effetti diretti sulla capacità sensoriali dei odontoceti e possibilmente compromettere i processi fisiologici essenziali. Questo studio sottolinea come potrebbe esserci effetti da varie fonti di rumore antropico, che possono causare cambiamenti fisici nei tessuti sensoriali dei cetacei e di altra fauna marinaMorfo-funcionalidad del canal auditivo externo en odontocetos. Si bien la contaminación acústica marina antropogénica es un asunto que preocupa a la comunidad científica y a la sociedad en general, existe un conocimiento limitado sobre cómo los animales marinos, en particular los cetáceos, perciben su entorno a través de los sonidos. Los odontocetos, como todos los cetáceos, presentan una serie de asombrosas adaptaciones evolutivas morfológicas y fisiológicas. Entre ellas, una de las más notables se encuentra en la compleja configuración del aparato auditivo. El canal auditivo externo, aunque no se considera que tenga un rol directo en el proceso de recepción del sonido, también muestra una sorprendente adaptación al medio marino, mostrando muchas estructuras activas y una compleja inervación periférica. No obstante, los conocimientos básicos sobre su morfología no son concluyentes por el momento. Este estudio tuvo como objetivo la descripción fundamental de la morfología del conducto auditivo externo en diversos odontocetos, asociándola a sus capacidades sensoriales a través de descripciones morfológicas en profundidad de su forma y curso, lumen y contenido, epitelio, glándulas, tejido linfoide, vascularización, inervación, tejido muscular, cartílago, tejido adiposo y conectivo. Además, se prestó especial atención a la identificación y caracterización morfológica de las formaciones sensoriales asociadas al canal auditivo externo, y se hace una comparación con el conducto auditivo externo de cetartiodáctilos terrestres, con el fin de alcanzar una comprensión integral de las capacidades sensoriales de los cetáceos. Se recolectaron muestras post-mortem durante necropsias de odontocetos salvajes, en un esfuerzo de colaboración internacional. Los tejidos se inspeccionaron macroscópicamente y se sometieron a estudios microscópicos, incluyendo análisis inmunohistoquímicos con anticuerpos específicos para tejido nervioso, diversas técnicas histoquímicas, investigación ultraestructural mediante microscopía electrónica de transmisión y reconstrucción 3D a partir de cortes histológicos del canal auditivo y tejidos asociados. La estructura sinuosa del canal auditivo externo reveló un órgano complejo cuya función fisiológica se refleja en sus delicadas estructuras anatómicas, la morfología de las cuales se discuten en detalle. Sorprendentemente, la inervación mostró un extenso plexo nervioso intramural con la presencia predominante de corpúsculos lamelares simples, similares a los corpúsculos de Pacini, aunque sin capa externa o cápsula. Se observaron diferencias en la conformación de los corpúsculos a lo largo del canal auditivo, desde una red que abarca completamente el conducto hasta una cresta de tejido nervioso que sobresale en el lumen. El trabajo desarrolla hipótesis relacionadas con la función del conducto auditivo externo y el sistema somatosensorial en odontocetos, teniendo en cuenta la importancia de la perspectiva de las modalidades sensoriales en el medio marino. Los resultados indican que el canal desempeña una función fisiológica importante, cuyo deterioro puede tener efectos directos sobre sus capacidades sensoriales y comprometer procesos fisiológicos esenciales. También cuestiona hasta qué punto las diversas fuentes de ruido antropogénico pueden tener un efecto adverso, ya que pueden provocar cambios físicos en los tejidos sensoriales en cetáceos y otra fauna marinaPostprint (published version

Neuroanatomy of the cetacean sensory systems

Cetaceans have undergone profound sensory adaptations in response to their aquatic environment during evolution. These adaptations are characterised by anatomo-functional changes in the classically defined sensory systems, shaping their neuroanatomy accordingly. This review offers a concise and up-to-date overview of our current understanding of the neuroanatomy associated with cetacean sensory systems. It encompasses a wide spectrum, ranging from the peripheral sensory cells responsible for detecting environmental cues, to the intricate structures within the central nervous system that process and interpret sensory information. Despite considerable progress in this field, numerous knowledge gaps persist, impeding a comprehensive and integrated understanding of their sensory adaptations, and through them, of their sensory perspective. By synthesising recent advances in neuroanatomical research, this review aims to shed light on the intricate sensory alterations that differentiate cetaceans from other mammals and allow them to thrive in the marine environment. Furthermore, it highlights pertinent knowledge gaps and invites future investigations to deepen our understanding of the complex processes in cetacean sensory ecology and anatomy, physiology and pathology in the scope of conservation biology.Peer ReviewedPostprint (published version

Acoustics in water: synergies with marine biology

This paper presents some of the bioacoustics related analysis that was performed on the ANTARES data, focussing on the year 2014. The data was processed for sperm whale, dolphin and shipping presence and grouped by hour of the day. It seemed that dolphins were more socially active during the day and foraging during the night. Sperm whales were mostly foraging during the day, but they may have been moving to other areas during the night. The most intense shipping noise came from a ferry that passed the platform twice a day. Although beaked whales were expected to be present in the area, so far their biosonar signal has not been conclusively found.Postprint (published version

Arctic anthropogenic sound contributions from seismic surveys during summer 2013

Statoil deployed three acoustic recorders from fall 2013 to 2014 in the Arctic region as part of a broad scientific campaign. One recorder was installed in the Barentsz Sea south-east of Spitsbergen. Two other recorders were installed in the Greenland Sea north-east of Greenland. All recorders were operating at a duty cycle of 2 min on and 30 min off, sampling at 39,062 Hz and recording in 24 bits. The Greenland recorders both captured air gun surveys performed during the summer months of 2013, allowing to estimate the transmission loss in the Arctic over long ranges. This paper presents “log(R)” transmission loss curves for these scenarios that can help assessing the acoustic shipping impact for future expeditions.Postprint (published version

Artificial sound impact could put at risk hermit crabs and their symbiont anemones

The sea anemone Calliactis parasitica, which is found in the East Atlantic (Portugal to Senegal) and the Mediterranean Sea, forms a symbiotic relationship with the red hermit crab, Dardanus calidus, in which the anemone provides protection from predators such as the octopus while it gains mobility, and possibly food scraps, from the hermit crab. Acoustic pollution is recognised by the scientific community as a growing threat to ocean inhabitants. Recent findings on marine invertebrates showed that exposure to artificial sound had direct behavioural, physiological and ultrastructural consequences. In this study we assess the impact of artificial sound (received level 157 ± 5 dB re 1 µPa2 with peak levels up to 175 dB re 1 µPa2) on the red hermit crab and its symbiotic sea anemone. Scanning electron microscopy analyses revealed lesions in the statocyst of the red hermit crab and in the tentacle sensory epithelia of its anemone when exposed to low-intensity, low-frequency sounds. These ultrastructural changes under situations of acoustic stress in symbiotic partners belonging to different phyla is a new issue that may limit their survival capacity, and a new challenge in assessing the effects of acoustic disturbance in the oceanic ecosystem. Despite the lesions found in the red hermit crab, its righting reflex time was not as strongly affected showing only an increase in the range of righting times. Given that low-frequency sound levels in the ocean are increasing and that reliable bioacoustic data on invertebrates is very scarce, in light of the results of the present study, we argue that anthropogenic sound effects on invertebrates species may have direct consequences in the entire ecosystem.Author J.-M. Fortuño acknowledges the institutional support of the “Severo Ochoa Centre of Excellence” accreditation (CEX 2019-000928-S).Peer ReviewedPostprint (published version

Morphological evidence for the sensitivity of the ear canal of odontocetes as shown by immunohistochemistry and transmission electron microscopy

The function of the external ear canal in cetaceans is still under debate and its morphology is largely unknown. Immunohistochemical (IHC) analyses using antibodies specific for nervous tissue (anti-S100, anti-NSE, anti-NF, and anti-PGP 9.5), together with transmission electron microscopy (TEM) and various histological techniques, were carried out to investigate the peripheral nervous system of the ear canals of several species of toothed whales and terrestrial Cetartiodactyla. This study highlights the innervation of the ear canal with the presence of lamellar corpuscles over its entire course, and their absence in all studied terrestrial mammals. Each corpuscle consisted of a central axon, surrounded by lamellae of Schwann receptor cells, surrounded by a thin cellular layer, as shown by IHC and TEM. These findings indicate that the corpuscles are mechanoreceptors that resemble the inner core of Pacinian corpuscles without capsule or outer core, and were labelled as simple lamellar corpuscles. They form part of a sensory system that may represent a unique phylogenetic feature of cetaceans, and an evolutionary adaptation to life in the marine environment. Although the exact function of the ear canal is not fully clear, we provide essential knowledge and a preliminary hypothetical deviation on its function as a unique sensory organ.Peer ReviewedPostprint (published version

Marine mammal acoustic detections in the Greenland and Barents Sea, 2013 – 2014 seasons

While the Greenland and Barents Seas are known habitats for several cetacean and pinniped species there is a lack of long-term monitoring data in this rapidly changing environment. Moreover, little is known of the ambient soundscapes, and increasing of-shore anthropogenic activities can infuence the ecosystem and marine life. Baseline acoustic data is needed to better assess current and future soundscape and ecosystem conditions. The analysis of a year of continuous data from three passive acoustic monitoring devices revealed species-dependent seasonal and spatial variation of a large variety of marine mammals in the Greenland and Barents Seas. Sampling rates were 39 and 78kHz in the respective locations, and all systems were operational at a duty cycle of 2min on, 30min of. The research presents a description of cetacean and pinniped acoustic detections along with a variety of unknown low-frequency tonal sounds, and ambient sound level measurements that fall within the scope of the European Marine Strategy Framework (MSFD). The presented data shows the importance of monitoring Arctic underwater biodiversity for assessing the ecological changes under the scope of climate changePeer ReviewedPostprint (published version

The Odontocete Ear Canal-Associated Lymphoid Tissue (ECALT) and Lymph Nodes: Morphological and Pathological Description with Immuno-Phenotypic Characterisation

A changing marine environment with emerging natural and anthropogenic stressors challenges the marine mammal immune system. The skin and adnexa form a first protective barrier in the immune response, although this is still relatively understudied in cetaceans. The cellular and tissue morphology of the nodular and diffuse lymphoid tissue are not fully charted and the physiological responses are not yet completely understood. The odontocete's external ear canal has a complex relationship with the external environment, with an artificial lumen rendering the inside of the canal a relatively secluded environment. In this work, we studied the odontocete ear canal-associated lymphoid tissue (ECALT) by histo- and immunohistochemistry (HC, IHC) with anti-CD3, anti-CD20, anti-Iba-1, anti-HLA-DR, and anti-vimentin antibodies. The ECALT cellular composition consists mainly of B-lymphocytes with the occasional presence of T-lymphocytes and the dispersed distribution of the macrophages. In cases of activation, the cellular reaction showed a similar pattern with the occasional presence of T-cells, plasma cells, and neutrophils. Nodular lymphoid tissue was generally in line with the description in other odontocetes, although with abundant erythrocytes throughout the entire organ. This study contributes to the understanding of the cellular composition of diffuse and nodular lymphoid tissue in several species of odontocetes, and in association with inflammation of the external ear canal

Preliminary findings on the peripheral nervous system of the external ear in odontocetes

Cetacean mass strandings have been associated with exposure to high-energy anthropogenic noise, and the post-mortem investigations indicate pathologies related to such events. Although these kinds of exposure can trigger behavioural and physiological changes, the direct impact a low frequency and high intensity noise exposure can have on the physical tissues is still unclear, particularly regarding the acoustic pathways. Odontocetes can perceive sounds through their mandible and the associated acoustic fat bodies, which are in physical contact with the tympanic bulla and the ear bones. However, the exact pathway of how sound waves reach the inner ear is still under debate. Moreover, the involved tissues have been described in only few species, and as there are big interspecific differences in the acoustic spectrum, this constitutes interspecific morphological differences along the sound production and reception pathways. Furthermore, the function of associated tissues such as the external ear canal is unknown. Even if there is a general consensus amongst the scientific community that cetaceans could suffer from acoustic trauma after sound exposure, very scarce data is currently available to confirm this, especially during mass stranding events. Here, we will present the first stages of this project which involves the strategic partnerships between multiple international institutions involved in the assessment of the health status of vibration sensitive tissues in the head of small odontocetes. Furthermore, we will open up possibilities for more extended collaborations, and we will present preliminary results on the functional morphology of the peripheral nervous system related to the mentioned tissues