Blubber gene expression and cortisol concentrations reveal changing physiological stress in a Southern ocean sentinel species.

The health of migratory eastern Australian humpback whales (Megaptera novaeangliae) can reflect the condition of their remote polar foraging environments. This study used gene expression (LEP, LEPR, ADIQ, AhR, TNF-α, HSP-70), blubber hormone concentrations (cortisol, testosterone), and photogrammetric body condition to assess this sentinel species during a period of unprecedented changes to anthropogenic activity and natural processes. The results revealed higher cortisol concentrations in 2020 compared to 2021, suggesting a decline in physiological stress between years. Additionally, metabolic transcripts LEPR, and AhR, which is also linked to xenobiotic metabolism, were upregulated during the 2020 southbound migration. These differences suggest that one or more environmental stressors were reduced between 2020 and 2021, with upregulated AhR possibly indicating a Southern Ocean pollutant declined between the years. This research confirms a Southern Ocean-wide decrease in whale stress during the study period and informs efforts to identify key stressors on Antarctic marine ecosystems.

A novel RT-qPCR health assay reveals differential expression of stress and immunoregulatory genes between the seasonal migrations of humpback whales (Megaptera novaeangliae).

Health information is essential for the conservation management of whale species. However, assessing the health of free-ranging whales is challenging as samples are primarily limited to skin and blubber tissue. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers a method to measure health from blubber RNA, providing insights into energetic status, stress and immune activity. To identify changes in health, natural differences in baseline gene expression linked to an individual's sex, reproductive status and life-history stage must first be quantified. This study aimed to establish baseline gene expression indices of health in migrating humpback whales (Megaptera novaeangliae). To do this, we developed an assay to quantify seven health-related gene transcripts (Leptin, Leptin Receptor, Adiponectin, Aryl Hydrocarbon Receptor, Tumour Necrosis Factor-α, Interleukin-6, Heat Shock Protein-70) and used Bayesian mixed effect models to assess differential baseline expression based on sex, lactation status and migration stage (northbound to and southbound from the annual breeding grounds). Results showed no significant contribution of sex to differential baseline expression. However, lactating individuals exhibited downregulated AhR and HSP-70 compared to non-lactating conspecifics. Additionally, southbound individuals demonstrated significantly upregulated HSP-70 and downregulated TNF-alpha, suggesting a relationship between these inflammation-linked transcripts and migratory fasting. Our results suggest that baseline differences due to migratory stage and lactation status should be considered in health applications of this assay. Future monitoring efforts can use our baseline measurements to better understand how gene expression is tied to population-level impacts, such as reduced prey availability or migratory stressors.

A mammalian messenger RNA sex determination method from humpback whale (Megaptera novaeangliae) blubber biopsies.

The large size of free-ranging mysticetes, such as humpback whales (Megaptera novaeangliae), make capture and release health assessments unfeasible for conservation research. However, individual energetic condition or reproductive health may be assessed from the gene expression of remotely biopsied tissue. To do this, researchers must reliably extract RNA and interpret gene expression measurements within the context of an individual's sex. Here, we outline an RNA extraction protocol from blubber tissue and describe a novel mammalian RNA sex determination method. Our method consists of a duplex reverse transcription-quantitative (real-time) polymerase chain reaction (RT-qPCR) with primer sets for a control gene (ACTB) and the X-chromosome inactivation gene (XIST). Products of each RT-qPCR had distinct melting temperature profiles based on the presence (female) or absence (male) of the XIST transcript. Using high-resolution melt analysis, reactions were sorted into one of two clusters (male/female) based on their melting profiles. We validated the XIST method by comparing results with a standard DNA-based method. With adequate quantities of RNA (minimum of approx. 9 ng µl-1), the XIST sex determination method shows 100% agreement with traditional DNA sex determination. Using the XIST method, future cetacean health studies can interpret gene expression within the context of an individual's sex, all from a single extraction.

Song complexity is maintained during inter-population cultural transmission of humpback whale songs.

Among animal species, the songs of male humpback whales (Megaptera novaeangliae) are a rare example of social learning between entire populations. Understanding fine-scale similarity in song patterns and structural features will better clarify how accurately songs are learned during inter-population transmission. Here, six distinct song types (2009-2015) transmitted from the east Australian to New Caledonian populations were quantitatively analysed using fine-scale song features. Results found that New Caledonian whales learned each song type with high accuracy regardless of the pattern's complexity. However, there were rare instances of themes (stereotyped patterns of sound units) only sung by a single population. These occurred more often in progressively changing 'evolutionary' songs compared to rapidly changing 'revolutionary' songs. Our results suggest that populations do not need to reduce complexity to accurately learn song patterns. Populations may also incorporate changes and embellishments into songs in the form of themes which are suggested to be learnt as distinct segments. Maintaining complex song patterns with such accuracy suggests significant acoustic contact, supporting the hypothesis that song learning may occur on shared feeding grounds or migration routes. This study improves the understanding of inter-population mechanisms for large-scale cultural transmission in animals.

Humpback whale adult females and calves balance acoustic contact with vocal crypsis during periods of increased separation.

Acoustic communication is important for animals with dependent young, particularly when they are spatially separated. Maternal humpback whales (Megaptera novaeangliae) use acoustic calling to help minimize the risk of separation from their young calves during migration. These pairs also use acoustic crypsis to minimize detection by males. How they balance a restricted active space with the need to maintain acoustic contact during periods of separation is not yet understood. Here, we analyzed movement metrics of tagged adult female-calf pairs during migration to identify two behavioral states, "resting/milling" and "travelling." When travelling, these pairs dived synchronously and exhibited little to no spatial separation. Alternatively, adult females had significantly longer dive durations (p < .01) when resting, and while they spent prolonged times at depth, calves would surface several times independently. This demonstrated that these pairs are frequently separated during periods of rest. We then determined whether the call rates and acoustic levels of these pairs increased with more frequent separation, finding that both adult females and calves significantly increased their call rates, but not levels, when resting. We also found that adult female-calf pairs have a restricted active space, with less than 15% of calls estimated to be detectable beyond 2 km. However, as with call level, detection distance did not differ significantly between the two behavioral states. In summary, adult female-calf pairs maintain successful communication during periods of separation by calling more frequently rather than by producing louder calls. This strategy aids in maintaining acoustic contact while simultaneously limiting detectability by conspecifics.

Fuzzy clustering as a tool to differentiate between discrete and graded call types.

Animals may communicate potential information to conspecifics using stereotyped "discrete" calls and variable "graded" calls. However, animal vocal research often centers on identifying the number of call types in a repertoire rather than quantifying the amount of gradation. Here, fuzzy clustering was applied to the social call repertoire of a species with a complex communication system, the humpback whale (Megaptera novaeangliae). Of 26 call types, 6 were classified as discrete, 7 as graded, and 13 as intermediate. These results indicate that humpback whales have a graded call repertoire, and fuzzy clustering may be a useful approach to investigate this variability.

Ships and air guns reduce social interactions in humpback whales at greater ranges than other behavioral impacts.

Understanding the interactions between human activity in the ocean and marine mammals is a fundamental step to developing responsible mitigation measures and informing policy. Here, the response of migrating humpback whales to vessels towing seismic air gun arrays (on or off) was quantified as a reduction in their likelihood of socially interacting (joining together). Groups were significantly less likely to participate in a joining interaction in the presence of a vessel, regardless of whether or not the air guns were active. This reduction was especially pronounced in groups within a social environment that favored joining, that is, when singing whales or other groups were nearby. Seismic survey mitigation practices are designed primarily to prevent damage to whales' hearing from close-by sources. Here, we found potentially detrimental behavioral changes at much greater ranges, and much lower received levels, than those used for current mitigation recommendations.

Blubber cortisol levels in humpback whales (Megaptera novaeangliae): A measure of physiological stress without effects from sampling.

Baleen whales are vulnerable to environmental impacts due to low fecundity, capital breeding strategies, and their reliance on a large amount of prey resources over large spatial scales. There has been growing interest in monitoring health and physiological stress in these species but, to date, few measures have been validated. The purpose of this study was to examine whether blubber cortisol could be used as a measure of physiological stress in humpback whales. Cortisol concentrations were initially compared between live, presumably 'healthy' whales (n = 187) and deceased whales (n = 35), which had died after stranding or entanglement, or washed ashore as a carcass. Deceased whales were found to have significantly higher cortisol levels (mean ± SD; 5.47 ± 4.52 ng/g) than live whales (0.51 ± 0.14 ng/g; p < 0.001), particularly for those animals that had experienced prolonged trauma (e.g. stranding) prior to death. Blubber cortisol levels in live whales were then examined for evidence of life history-related, seasonal, or sampling-related effects. Life history group and sampling-related factors, such as encounter time and the number of biopsy sampling attempts per animal, were found to be poor predictors of blubber cortisol levels in live whales. In contrast, blubber cortisol levels varied seasonally, with whales migrating north towards the breeding grounds in winter having significantly higher levels (0.54 ± 0.21 ng/g, p = 0.016) than those migrating south towards the feeding grounds in spring (0.48 ± 1.23 ng/g). These differences could be due to additional socio-physiological stress experienced by whales during peaks in breeding activity. Overall, blubber cortisol appears to be a suitable measure of chronic physiological stress in humpback whales.

Network analysis reveals underlying syntactic features in a vocally learnt mammalian display, humpback whale song.

Vocal communication systems have a set of rules that govern the arrangement of acoustic signals, broadly defined as 'syntax'. However, there is a limited understanding of potentially shared or analogous rules across vocal displays in different taxa. Recent work on songbirds has investigated syntax using network-based modelling. This technique quantifies features such as connectivity (adjacent signals in a sequence) and recurring patterns. Here, we apply network-based modelling to the complex, hierarchically structured songs of humpback whales (Megaptera novaeangliae) from east Australia. Given the song's annual evolving pattern and the cultural conformity of males within a population, network modelling captured the patterns of multiple song types over 13 consecutive years. Song arrangements in each year displayed clear 'small-world' network structure, characterized by clusters of highly connected sounds. Transitions between these connected sounds further suggested a combination of both structural stability and variability. Small-world network structure within humpback songs may facilitate the characteristic and persistent vocal learning observed. Similar small-world structures and transition patterns are found in several birdsong displays, indicating common syntactic patterns among vocal learning in multiple taxa. Understanding the syntactic rules governing vocal displays in multiple, independently evolving lineages may indicate what rules or structural features are important to the evolution of complex communication, including human language.

The effects of vessel noise on the communication network of humpback whales.

Humpback whales rely on acoustic communication to mediate social interactions. The distance to which these social signals propagate from the signaller defines its communication space, and therefore communication network (number of potential receivers). As humpback whales migrate along populated coastlines, they are likely to encounter noise from vessel traffic which will mask their social signals. Since no empirical data exist on baleen whale hearing, the consequences of this are usually assumed, being the modelled reduction in their communication space. Here, the communication space and network of migrating humpback whales was compared in increasing wind-dominated and vessel-dominated noise. Behavioural data on their social interactions were then used to inform these models. In typical wind noise, a signaller's communication space was estimated to extend to 4 km, which agreed with the maximum separation distance between groups that socially interacted. An increase in vessel noise reduced the modelled communication area, along with a significant reduction in group social interactions, probably due to a reduction in their communication network. However, signal masking did not fully explain this change in social behaviour, implying there was also an additional effect of the physical presence of the vessel on signaller and receiver behaviour. Though these observed changes in communication space and social behaviour were likely to be short term and localized, an increase in vessel activity due to tourism and coastal population growth may cause more sustained changes along the humpback whale migration paths.

Source levels of humpback whales decrease with frequency suggesting an air-filled resonator is used in sound production.

Source level and frequency are important in determining how far an acoustic signal can travel. However, in some species these sound characteristics have been found to be biomechanically linked, and therefore cannot be modified independently to achieve optimal transmission. This study investigates the variability in source levels and their relationship with frequency in the songs of humpback whales (Megaptera novaeangliae). Songs were recorded off eastern Australia using a fixed hydrophone array deployed on the whales' migratory corridor. Singing whales were acoustically tracked. An empirical, frequency-dependent model was used to estimate transmission loss. Source levels and frequency were measured for 2408 song units from 19 singers. Source levels varied from 138 to 187 dB re 1 µPa at 1 m (root mean squared), while peak frequency ranged between 52 and 3877 Hz. Much of the variability in source levels was accounted for by differences between the unit types, with mean source levels for each unit type varying by up to 17 dB. Source levels were negatively correlated with peak frequency and decreased by 2.3 dB per octave. The negative correlation between source levels and frequency is consistent with the presence of an air-filled resonator in the whales' sound production system.

Cultural revolutions reduce complexity in the songs of humpback whales.

Much evidence for non-human culture comes from vocally learned displays, such as the vocal dialects and song displays of birds and cetaceans. While many oscine birds use song complexity to assess male fitness, the role of complexity in humpback whale (Megaptera novaeangliae) song is uncertain owing to population-wide conformity to one song pattern. Although songs change gradually each year, the eastern Australian population also completely replaces their song every few years in cultural 'revolutions'. Revolutions involve learning large amounts of novel material introduced from the Western Australian population. We examined two measures of song structure, complexity and entropy, in the eastern Australian population over 13 consecutive years. These measures aimed to identify the role of complexity and information content in the vocal learning processes of humpback whales. Complexity was quantified at two hierarchical levels: the entire sequence of individual sound 'units' and the stereotyped arrangements of units which comprise a 'theme'. Complexity increased as songs evolved over time but decreased when revolutions occurred. No correlation between complexity and entropy estimates suggests that changes to complexity may represent embellishment to the song which could allow males to stand out amidst population-wide conformity. The consistent reduction in complexity during song revolutions suggests a potential limit to the social learning capacity of novel material in humpback whales.

The communication space of humpback whale social sounds in wind-dominated noise.

In animal social networks, a large acoustic communication space tends to involve complex networks. Signal masking may reduce this space, leading to detrimental effects on the animal's ability to obtain important social information. Humpback whales use acoustic social sounds (vocal sounds and surface-generated sounds from breaching or fin slapping) for within- and between-group communication. In this study, changes in various sound parameters (e.g., signal-above-noise and frequency content) of received humpback whale social sounds were statistically modeled against the combined effect of increasing wind-dominated noise and distance from the source (whale) to produce masking models. Behavioral data on vocalizing groups were also used to inform these models. The acoustic communication space, in this shallow water (<50 m) environment, extended to approximately 4 km from the signaler in median wind noise. However, the majority of behavioral interactions occurred within 2 km of the signaler. Surface-generated signals propagated better and likely function to maintain this space in higher wind noise. This study provides a basic wind-noise masking model for social communication signals in humpback whales which can be updated as more information on humpback auditory capabilities, and potential masking effects of anthropogenic noise sources, becomes available.

A behavioural dose-response model for migrating humpback whales and seismic air gun noise.

The behavioural responses of migrating humpback whales to an air gun, a small clustered seismic array and a commercial array were used to develop a dose-response model, accounting for the presence of the vessel, array towpath relative to the migration and social and environmental parameters. Whale groups were more likely to show an avoidance response (increasing their distance from the source) when the received sound exposure level was over 130 dB re 1 µPa2·s and they were within 4 km of the source. The 50% probability of response occurred where received levels were 150-155 dB re 1 µPa2·s and they were within 2.5 km of the source. A small number of whales moving rapidly close to the source vessel did not exhibit an avoidance response at the highest received levels (160-170 dB re 1 µPa2·s) meaning it was not possible to estimate the maximum response threshold.

Stereotypic and complex phrase types provide structural evidence for a multi-message display in humpback whales (Megaptera novaeangliae).

Male humpback whales produce a mating display called "song." Behavioral studies indicate song has inter- and/or intra-sexual functionality, suggesting song may be a multi-message display. Multi-message displays often include stereotypic components that convey group membership for mate attraction and/or male-male interactions, and complex components that convey individual quality for courtship. Humpback whale song contains sounds ("units") arranged into sequences ("phrases"). Repetitions of a specific phrase create a "theme." Within a theme, imperfect phrase repetitions ("phrase variants") create variability among phrases of the same type ("phrase type"). The hypothesis that song contains stereotypic and complex phrase types, structural characteristics consistent with a multi-message display, is investigated using recordings of 17 east Australian males (8:2004, 9:2011). Phrase types are categorized as stereotypic or complex using number of unit types, number of phrase variants, and the proportion of phrases that is unique to an individual versus shared amongst males. Unit types are determined using self-organizing maps. Phrase variants are determined by Levenshtein distances between phrases. Stereotypic phrase types have smaller numbers of unit types and shared phrase variants. Complex phrase types have larger numbers of unit types and unique phrase variants. This study supports the hypothesis that song could be a multi-message display.

The behavioural response of migrating humpback whales to a full seismic airgun array.

Despite concerns on the effects of noise from seismic survey airguns on marine organisms, there remains uncertainty as to the biological significance of any response. This study quantifies and interprets the response of migrating humpback whales (Megaptera novaeangliae) to a 3130 in3 (51.3l) commercial airgun array. We compare the behavioural responses to active trials (array operational; n = 34 whale groups), with responses to control trials (source vessel towing the array while silent; n = 33) and baseline studies of normal behaviour in the absence of the vessel (n = 85). No abnormal behaviours were recorded during the trials. However, in response to the active seismic array and the controls, the whales displayed changes in behaviour. Changes in respiration rate were of a similar magnitude to changes in baseline groups being joined by other animals suggesting any change group energetics was within their behavioural repertoire. However, the reduced progression southwards in response to the active treatments, for some cohorts, was below typical migratory speeds. This response was more likely to occur within 4 km from the array at received levels over 135 dB re 1 µPa2s.

Using self-organizing maps to classify humpback whale song units and quantify their similarity.

Classification of vocal signals can be undertaken using a wide variety of qualitative and quantitative techniques. Using east Australian humpback whale song from 2002 to 2014, a subset of vocal signals was acoustically measured and then classified using a Self-Organizing Map (SOM). The SOM created (1) an acoustic dictionary of units representing the song's repertoire, and (2) Cartesian distance measurements among all unit types (SOM nodes). Utilizing the SOM dictionary as a guide, additional song recordings from east Australia were rapidly (manually) transcribed. To assess the similarity in song sequences, the Cartesian distance output from the SOM was applied in Levenshtein distance similarity analyses as a weighting factor to better incorporate unit similarity in the calculation (previously a qualitative process). SOMs provide a more robust and repeatable means of categorizing acoustic signals along with a clear quantitative measurement of sound type similarity based on acoustic features. This method can be utilized for a wide variety of acoustic databases especially those containing very large datasets and can be applied across the vocalization research community to help address concerns surrounding inconsistency in manual classification.

Changes in humpback whale singing behavior with abundance: Implications for the development of acoustic surveys of cetaceans.

Acoustic surveys of vocal animals can have significant advantages over visual surveys, particularly for marine mammals. For acoustic density estimates to be viable, however, the vocal output of the animals surveyed needs to be determined under a range of conditions and shown to be a robust predictor of abundance. In this study, the songs of humpback whales, one of the most vocal and best studied species of marine mammals, were tested as predictors of abundance. Two acoustic metrics, the number of singing whales and amount of songs produced, were compared with the number of whales seen traversing a study site on the eastern coast of Australia over an 18 year period. Although there were predictive relationships between both metrics and numbers of passing whales, these relationships changed significantly as the population grew in size. The proportion of passing whales that sang decreased as the population increased. Singing in humpback whales, therefore, is a poor predictor even of relative abundance and illustrates the caution required when developing acoustic survey techniques particularly when using social vocalizations.

Determining the behavioural dose-response relationship of marine mammals to air gun noise and source proximity.

The effect of various anthropogenic sources of noise (e.g. sonar, seismic surveys) on the behaviour of marine mammals is sometimes quantified as a dose-response relationship, where the probability of an animal behaviourally 'responding' (e.g. avoiding the source) increases with 'dose' (or received level of noise). To do this, however, requires a definition of a 'significant' response (avoidance), which can be difficult to quantify. There is also the potential that the animal 'avoids' not only the source of noise but also the vessel operating the source, complicating the relationship. The proximity of the source is an important variable to consider in the response, yet difficult to account for given that received level and proximity are highly correlated. This study used the behavioural response of humpback whales to noise from two different air gun arrays (20 and 140 cubic inch air gun array) to determine whether a dose-response relationship existed. To do this, a measure of avoidance of the source was developed, and the magnitude (rather than probability) of this response was tested against dose. The proximity to the source, and the vessel itself, was included within the one-analysis model. Humpback whales were more likely to avoid the air gun arrays (but not the controls) within 3 km of the source at levels over 140 re. 1 µPa2 s-1, meaning that both the proximity and the received level were important factors and the relationship between dose (received level) and response is not a simple one.

Potential motivational information encoded within humpback whale non-song vocal sounds.

Acoustic signals in terrestrial animals follow motivational-structural rules to inform receivers of the signaler's motivational state, valence and level of arousal. Low-frequency "harsh" signals are produced in aggressive contexts, whereas high-frequency tonal sounds are produced in fearful/appeasement contexts. Using the non-song social call catalogue of humpback whales (Megaptera novaeangliae), this study tested for potential motivational-structural rules within the call catalogue of a baleen whale species. A total of 32 groups within different social contexts (ranging from stable, low arousal groups, such as a female with her calf, to affiliating, higher arousal, groups containing multiple males competing for access to the central female) were visually and acoustically tracked as they migrated southwards along the eastern coast of Australia. Social calls separated into four main cluster types, with signal structures in two categories consistent with "aggressive" signals and, "fearful/appeasement" signals in terrestrial animals. The group's use of signals within these clusters matched their context in that presumed low arousal non-affiliating groups almost exclusively used "low-arousal" signals (a cluster of low frequency unmodulated or upsweep sounds). Affiliating groups used a higher proportion of an intermediate cluster of signal types deemed "higher arousal" signals and groups containing three or more adults used a higher proportion of "aggressive" signal types.