The social role of sound production and hearing in a highly vocal teleost fish, Halobatrachus didactylus

Tese de doutoramento, Biologia (Ecofisiologia), Universidade de Lisboa, Faculdade de Ciências, 2011The existence of individual vocal signatures that can provide scope for mate choice has been scarcely investigated in fish. The relation between acoustic signalling and reproductive success has been studied in various taxa but likewise remains unclear in this taxon. Ultimately, studies that examine sound production and auditory reception, within a comparative perspective across species, can provide insights into the evolution of communication systems. This thesis focused on the social role of acoustic signaling and hearing in the Lusitanian toadfish Halobatrachis didactylus (Batrachoididae), which relies heavily on acoustic communication. The goals were: (1) determine whether male advertising boatwhistles can potentially provide individual recognition and if the sonic muscle variability is related with males‟ quality; verify the influence of vocal behaviour in the reproductive success; test the possible function of boatwhistles in nest defence; (2) compare auditory sensitivity across seasons and between sexes; characterise the representation of vocalizations in the auditory system; and (3) analyze the development of acoustic communication. Boatwhistles were different between males and the dominant frequency and frequency modulation were the parameters that best discriminate individuals. Sonic muscle variability was best explained by the body length and condition, suggesting that vocal output can inform about sender‟s quality. Reproductive success was significantly influenced by males‟ calling rate and calling effort. Besides mate attraction, boatwhistles were used during active territorial defence. The inner ear saccule was well suited to detect conspecific vocalizations throughout the year and sensitivity was not seasonally plastic or sexually dimorphic. The auditory system was capable of resolving fine vocal features probably important in communication. Moreover, the ability to communicate acoustically might be absent in early developmental stages and initiates when juveniles start generating higher amplitude calls with lower dominant frequencies. Finally, the development of the peripheral auditory system seems to parallels vocal differentiation in this species.A relação entre a emissão de sinais acústicos e o sucesso reprodutor é importante para compreender a evolução de sistemas que se baseiam fortemente na comunicação vocal e tem sido amplamente estudada em vários taxa, com excepção dos peixes. Por outro lado, a existência de padrões vocais individuais, potencialmente usados no processo de selecção sexual, tem sido pouco investigada neste taxon, apesar de peixes da família Batrachoididae revelarem diferenças vocais individuais relativamente elaboradas. No entanto, a função desta plasticidade vocal potencialmente mediadora do reconhecimento individual permanece muito pouco compreendida. Para além disso, estudos que examinam a produção de sons e a recepção auditiva, numa perspectiva comparativa entre espécies evolutivamente próximas, podem fornecer informações importantes para a compreensão da diversidade e evolução dos sistemas de comunicação. A presente tese centrou-se no estudo do papel social da produção de sons e nas capacidades auditivas do xarroco Halobatrachus didactylus (Teleostei: Batrachoididae). Esta espécie baseia-se fortemente na comunicação acústica para a atracção de parceiros sexuais na época de reprodução e para a resolução de interacções agonísticas, exibindo um repertório acústico invulgarmente complexo para peixes. O xarroco foi usado como modelo de estudo para investigar os seguintes objectivos: (1) determinar se vocalizações dos machos emitidas para atrair as fêmeas (sirenes) possibilitam o reconhecimento individual e podem ser potencialmente usadas pelas fêmeas para avaliar a qualidade do parceiro sexual; verificar a influência do comportamento vocal no sucesso reprodutor (número de ovos); testar a possível função dupla das sirenes em contextos de atracção sexual e agonísticos e verificar eventuais modificações dos seus parâmetros acústicos com o contexto social; (2) determinar a sensibilidade auditiva através do registo dos potenciais auditivos ao nível das células sensoriais do sáculo (principal órgão auditivo em teleósteos) e verificar eventuais diferenças sazonais e inter-sexuais; avaliar a representação de sons naturais no sistema auditivo, nomeadamente vocalizações conspecíficas e estímulos ecologicamente relevantes (i.e. sons de um potencial predador e de uma espécie simpátrica); (3) analisar o desenvolvimento da capacidade de produção de sons e da sensibilidade auditiva, i.e. do início da comunicação acústica; verificar se o desenvolvimento do sistema auditivo periférico (sensibilidade do sáculo) acompanha a diferenciação vocal. 5 A actividade vocal do xarroco foi registada em machos nidificantes numa zona intertidal no estuário do Tejo e, seguidamente, foram analisados os sons de atracção sexual (sirenes) relativamente a vários parâmetros acústicos. Todos os parâmetros analisados revelaram significativamente maior variabilidade inter-individual do que intra-individual. A frequência dominante e a modulação de frequência, seguidas do período entre pulsos, modulação de amplitude e duração total, foram as variáveis que melhor discriminaram diferentes machos. Estes resultados suportam a possibilidade de reconhecimento individual com base em características acústicas nesta espécie. Posteriormente, procedeu-se à quantificação da massa dos músculos sónicos intrínsecos à bexiga gasosa (aparelho produtor de som) em vários espécimes, machos e fêmeas, bem como à medição de outros parâmetros morfométricos. A variabilidade presente na massa dos músculos sónicos foi explicada principalmente pelo comprimento total e condição em ambos os sexos. Como a massa dos músculos está relacionada fortemente com o desempenho vocal, estes resultados sugerem que as vocalizações do xarroco podem informar sobre a qualidade do emissor, informação crítica em contexto reprodutor e agonístico. Finalmente, de forma a avaliar se o desempenho vocal condiciona o sucesso reprodutor, vários machos foram mantidos em condições semi-naturais no pico da época de reprodução em ninhos artificiais (que asseguraram a identidade dos espécimes monitorizados ao longo do estudo). A taxa máxima de canto e o esforço vocal (tempo despendido a cantar) foram os melhores preditores do sucesso reprodutor, avaliado pelo número de ovos presentes nos ninhos. Estes parâmetros vocais, por sua vez, encontraram-se correlacionados com o comprimento total e condição dos machos, indicando que o desempenho vocal condiciona fortemente o sucesso reprodutor e é informativo da qualidade do macho. As sirenes dos batracoidídeos foram descritas até à data como sinais emitidos pelos machos para atracção das fêmeas e usados na sinalização passiva de ninhos na época reprodutora. No entanto, observações prévias apontam para a sua ocorrência fora da época de reprodução. Assim sendo, elaborou-se um desenho experimental para verificar se estes sinais são produzidos igualmente em contexto de defesa territorial, que deverá existir todo o ano. As experiências incluíram a colocação de machos residentes num tanque experimental e a libertação de machos intrusos, tendo-se procedido ao registo comportamental das interacções visuais agonísticas e dos sinais acústicos emitidos. Os machos residentes, contrariamente aos intrusos, emitiram sirenes 6 agonísticas aquando da tentativa de ocupação dos ninhos por parte dos intrusos. As sirenes agonísticas foram emitidas em menor taxa, comparativamente com as sirenes de atracção sexual que são tipicamente emitidas em séries, com taxa mais constante e durante longos períodos de tempo. Ambos os sinais apresentaram duração e estrutura harmónica semelhantes, mas diferenças significativas na modulação de amplitude, frequência dominante e fundamental. Estas diferenças estão provavelmente relacionadas com a diferente taxa de emissão e com as exigências necessárias para a propagação dos sinais a distâncias diferentes do receptor. Uma vez que o xarroco utiliza fortemente o canal acústico durante interacções sociais e exibe um repertório vocal complexo, é de prever que o seu sistema auditivo seja capaz de codificar características finas das suas vocalizações. No entanto, esta espécie não possui estruturas morfológicas para optimizar a audição. De forma a avaliar as capacidades auditivas do xarroco, foram registados os potenciais auditivos em populações de células sensoriais da mácula no sáculo do ouvido interno, em machos e fêmeas, dentro e fora da época de reprodução. Os audiogramas obtidos indicaram maior sensibilidade a baixas frequências inferiores a 205 Hz, onde se encontra a maior parte da energia dos sons conspecíficos. Também foi verificada a existência de elevada sensibilidade a frequências muito baixas como 15 Hz, o que sugere uma sobreposição da sensibilidade auditiva com a sensibilidade da linha lateral. Não foram encontradas diferenças auditivas sazonais e inter-sexuais. Estes resultados demonstram que o sistema auditivo periférico de fêmeas e machos do xarroco encontra-se bem adaptado para a detecção de vocalizações conspecíficas e de sons de frequências muito baixas durante todo o ano, o que certamente optimiza a detecção de predadores/presas e a presença de conspecíficos no meio natural. Contrariamente a outro batracoidídeo amplamente estudado (Porichthys notatus) que apenas revela actividade vocal no período reprodutor e cujas fêmeas sofrem alterações sensoriais para melhor detectarem os sons dos machos, H. didactylus comunica acusticamente todo o ano e possivelmente por isso a sua sensibilidade auditiva não sofre modificações sazonais. Por outro lado, de forma a avaliar até que ponto o xarroco é capaz de integrar a complexidade das suas vocalizações, registaram-se os potenciais auditivos evocados (somatório da resposta neural sincronizada evocada por estimulação acústica) em machos e fêmeas. O sistema auditivo do xarroco demonstrou ser capaz de codificar parâmetros finos das suas vocalizações, nomeadamente de representar com elevada precisão a duração e estrutura pulsada dos sinais conspecíficos e ainda de resolver a 7 modulação de amplitude e o conteúdo espectral das sirenes. Adicionalmente, verificouse que esta espécie é capaz de detectar outros estímulos ecologicamente relevantes, i.e. sons de um potencial predador como o golfinho-roaz corvineiro e sons de outro peixe simpátrico bastante vocal como a corvina. Com base na mesma técnica electrofisiológica (somatório dos potenciais auditivos evocados), efectuou-se a medição da sensibilidade auditiva em diferentes tamanhos de juvenis e em adultos, tendo sido também gravados os seus sons agonísticos (tamborilados). A comparação dos vários audiogramas e conteúdos espectrais das vocalizações, permitiu verificar que a capacidade para comunicar acusticamente surge em etapas iniciais do desenvolvimento possivelmente quando os juvenis (> 5 cm de comprimento standard, CS) começam a ser capazes de produzir sons de maior amplitude e com frequências dominantes mais baixas. Por último, através do registo dos potenciais auditivos no sáculo do ouvido interno, foi possível verificar que o sistema auditivo periférico ainda não se encontra completamente desenvolvido em espécimens com menos de 5 cm CS e que o aumento da sensibilidade auditiva é acompanhado pela diferenciação vocal (aumento do repertório acústico) que acompanha o crescimento. Juvenis com mais de 5 cm CS, contrariamente aos mais pequenos, exibem uma sensibilidade auditiva já idêntica à dos adultos, bem como o repertório vocal completo. Os trabalhos constituintes desta tese demonstram que o sistema comunicativo do xarroco é mais complexo do que inicialmente se encontrava descrito, sendo comparável nalguns aspectos à complexidade de sistemas vocais presente noutros taxa como anuros e aves. A demonstração da presença de assinaturas vocais, do papel fundamental do canto no sucesso reprodutor, da capacidade para codificar características complexas das vocalizações, do paralelismo entre o desenvolvimento dos sistemas auditivo e vocal num peixe teleósteo, constituiu certamente um avanço importante para a compreensão da diversidade e evolução dos sistemas de comunicação.Fundação para a Ciência e a Tecnologia (FCT,SFRH/BD/30491/2006

Development of vocalization, auditory sensitivity and acoustic communication in the Lusitanian toadfish Halobatrachus didactylus

The ontogenetic development of acoustic communication has so far only been investigated in one fish species. In order to determine whether detectability of conspecific sounds changes during growth in a species with limited hearing abilities (generalist), we investigated the development of auditory sensitivity and agonistic vocalizations in the Lusitanian toadfish Halobatrachus didactylus. Agonistic grunts were recorded, their sound pressure levels determined, and auditory sensitivities measured in five different size groups ranging from 3 to 32·cm standard length. Hearing thresholds were obtained using the auditory evoked potentials (AEP) recording technique. Dominant frequency, sound duration and number of pulses decreased, whereas pulse period and sound level increased with increasing fish size. The best hearing was below 300·Hz in all groups. Lower hearing sensitivity was found in the smallest juveniles at 100·Hz as well as at higher frequencies (800 and 1000·Hz). Comparisons between audiograms and sound spectra within the same-sized fish revealed that smaller juveniles would be barely able to detect agonistic grunts, while these vocalizations were clearly perceived by larger fish. In the latter, the main energy of sounds was found at the most sensitive frequencies. This study demonstrates that acoustic communication in the Lusitanian toadfish might be absent in early developmental stages and seems to start when juveniles are able to generate grunts of higher sound level and lower dominant frequency

Effects of ship noise on the detectability of communication signals in the Lusitanian toadfish

Underwater noise pollution is an increasing environmental problem which might affect communication, behaviour, fitness and consequently species’ survival. The most common anthropogenic noises in aquatic habitats derive from shipping. In the present study we investigated the implications of noise pollution from a ship on the sound detectability, namely of conspecific vocalizations in the Lusitanian toadfish, Halobatrachus didactylus. Ambient and ferry-boat noises were recorded in the Tagus River estuary (Portugal), as well as toadfish sounds, and their sound pressure levels determined. Hearing sensitivities were measured under quiet lab conditions and in the presence of these masking noises at levels encountered in the field, using the auditory evoked potentials (AEP) recording technique. The Lusitanian toadfish is a hearing generalist, with best hearing sensitivity at low frequencies between 50 and 200·Hz (below 100·dB re. 1· Pa). Under ambient noise conditions, hearing was only slightly masked at lower frequencies. In the presence of ship noise, auditory thresholds increased considerably, by up to 36·dB, at most frequencies tested. This is mainly because the main energies of ferry-boat noise were within the most sensitive hearing range of this species. Comparisons between masked audiograms and sound spectra of the toadfish’s mating and agonistic vocalizations revealed that ship noise decreased the ability to detect conspecific acoustic signals. This study provides the first evidence that fishes’ auditory sensitivity can be impaired by ship noise and that acoustic communication, which is essential during agonistic encounters and mate attraction, might be restricted in coastal environments altered by human activity

Vocal behavior during territorial intrusions in the lusitanian toadfish: Boatwhistles also function as territorial ‘keep-out’ signals

Male signals are frequently studied in a single behavioral context, but in some cases they may assist multiple functions, namely for both male– male competition and female mate choice. Boatwhistles are known as the mate attraction calls of toadfishes typically produced during the breeding season. However, recent observations with the Lusitanian toadfish Halobatrachus didactylus (Batrachoididae) indicate that the emission of boatwhistles is not restricted to this period, which suggests a function in other behavioral contexts such as agonistic territorial interactions. We experimentally manipulated the social context of toadfish males to investigate whether boatwhistles are produced during territorial defense, by introducing ‘intruders’ in an experimental tank containing nesting ‘resident’ males. Furthermore, we examined whether parental care (eggs in the nest) affected the behavioral responses of resident males during territorial defense. Resident males defended their shelters producing sounds, mostly boatwhistles, towards intruders. Parental males revealed higher aggression levels, exhibiting additional threatening and attack behaviors. Boatwhistles registered during agonistic events were compared with the mate advertising boatwhistles recorded from small aggregations of nesting males in a natural breeding intertidal area. Agonistic boatwhistles were produced in lower and variable calling rates comparing with the advertising ones that were typically emitted in long series of calls. Agonistic boatwhistles were similar in duration and frequency harmonic structure (with a middle tonal phase) to the advertising calls, but presented less amplitude modulation, and lower dominant and fundamental frequencies. These acoustic differences were probably related to differences in calling rates and broadcast demands associated to the distance to the intended receiver. We provide first evidence that, apart from attracting mates, the toadfish boatwhistles also function as active ‘keep-out’ signals during territorial defense

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: 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.Fundo para o Desenvolvimento das Ciências e da Tecnologia, Macau S.A.R.; Fundaçao para a Ciência e a Tecnologia (FCT); Royalty Research Fund, US

Relative growth of carcass tissues of goat kids from five breed Types finished on pasture or feedlot

The aim of this study was to evaluate the effect of cross breeding, finishing system, and gender on the relative growth of carcass tissues of dairy kids. Seventy eight kids (39 male and 39 female) from five breed types were used: Alpine; ½ Boer + ½ Alpine (½ BA); ½ Nubian + ½ Alpine (½ ANA); ¾ Boer + ¼ Alpine (¾ BA); and ½ Nubian + ¼ Boer + ¼ Alpine (TC). Kids were distributed into two finishing systems: in pasture with doe (FS1) and weaned in feedlot (FS2). Kids were slaughtered at a mean age of 128.4 ± 7.9 days and mean live weight of 22.07 kg. The mean weight of half carcasses was 5.09 kg. To determine allometric growth, we used the exponential equation Y= aXb. In the half carcass, muscle tissue showed comparatively early growth in group ½ BA, whereas fat tissue of animals in FS1 had relatively late growth. Females exhibited early growth of muscle tissue, while in males this tissue was intermediate. The ½ BA first-cross improved carcass characteristics by enhancing the growth of muscle tissue

Relative growth of carcass tissues of goat kids from five breed Types finished on pasture or feedlot

he aim of this study was to evaluate the effect of cross breeding, finishing system, and gender on the relative growth of carcass tissues of dairy kids. Seventy eight kids (39 male and 39 female) from five breed types were used: Alpine; ½ Boer + ½ Alpine (½ BA); ½ Nubian + ½ Alpine (½ ANA); ¾ Boer + ¼ Alpine (¾ BA); and ½ Nubian + ¼ Boer + ¼ Alpine (TC). Kids were distributed into two finishing systems: in pasture with doe (FS1) and weaned in feedlot (FS2). Kids were slaughtered at a mean age of 128.4 ± 7.9 days and mean live weight of 22.07 kg. The mean weight of half carcasses was 5.09 kg. To determine allometric growth, we used the exponential equation Y= aXb. In the half carcass, muscle tissue showed comparatively early growth in group ½ BA, whereas fat tissue of animals in FS1 had relatively late growth. Females exhibited early growth of muscle tissue, while in males this tissue was intermediate. The ½ BA first-cross improved carcass characteristics by enhancing the growth of muscle tissue

Variability in the sonic muscles of the lusitanian toadfish (Halobatrachus didactylus): Acoustic signals may reflect individual quality

Animal vocalizations are good examples of signals that have been shaped by sexual selection and often contribute to resolve contests or the choice of mates. We relate the mass of the sound-producing muscles of a highly vocal fish species, the Lusitanian toadfish (Halobatrachus didactylus (Bloch and Schneider, 1801)), with the sender’s physical features, such as body size, and reproductive and body condition. In this species, both sexes are known to emit sounds during agonistic interactions and males rely on their mate attraction vocalizations to reproduce. Sonic muscles were highly variable among males (CV = 40%) and females (CV = 33%) and showed sexual dimorphism. Regression analysis showed that variability in the sonic muscles was best explained by total length and fish condition in males and females. Liver mass in both genders, and the mass of the testes accessory glands, also explained sonic muscle variability. These variables explained 96% and 91% of the sonic muscle mass variability in males and females, respectively. As in teleost fishes sonic muscle mass correlates to particular sound acoustic features, we propose that in the Lusitanian toadfish sounds can inform the receiver about the sender’s quality, such as body size and condition, which are critical information in contests and mate choice

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

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

Yield, composition, and fatty acid profile of milk from Anglo Nubian goats fed a diet supplemented with vegetable oils

We aimed to evaluate the inclusion of three sources of vegetable oil in the diet of lactating goats on production in 120 days of lactation and the effect of these sources and lactation stage on fortnightly composition and fatty acid profile of goat milk at 20, 50, 80, and 110 days of lactation. A completely randomized design was adopted and 32 Anglo Nubian goats were used, distributed in four treatments: control diet and diets with inclusion of 30 g/kg of dry matter of diet of canola, sunflower, or soybean oil. The dairy production was 182.75 kg, and there was no difference for treatments. Among the constituents, only urea nitrogen was influenced by treatment and presented lower content for control treatment. Day of lactation had an effect on lactose. Defatted dry extract and somatic cell count had a quadratic effect with minimum values around 100 and 33 days of lactation, respectively. The content of urea nitrogen, also with a quadratic effect, was higher at 93 days of lactation. For protein, there was an interaction between treatments and period and, at the end of lactation, its content was increased. The inclusion of vegetable oils promoted reduction in total saturated fatty acids (SFA) and increased the total content of monounsaturated fatty acids (MUFA) and conjugated linoleic acid. The proportions MUFA:SFA and PUFA:SFA, the atherogenicity and thrombogenicity indexes, and the relation hypocholesterolemic fatty acids:hypercholesterolemic fatty acids improved with oil addition in animal diets. The addition of vegetable oil to diets for lactating goats improve the fatty acid profile with no impairment on milk production and composition, and the milk from early stages of lactation has better nutritional quality of the lipid fractio