The active space of sperm whale codas: inter-click information for intra-unit communication.

Sperm whales (Physeter macrocephalus) are social mega-predators who form stable matrilineal units that often associate within a larger vocal clan. Clan membership is defined by sharing a repertoire of coda types consisting of specific temporal spacings of multi-pulsed clicks. It has been hypothesized that codas communicate membership across socially segregated sympatric clans, but others propose that codas are primarily used for behavioral coordination and social cohesion within a closely spaced social unit. Here, we test these hypotheses by combining measures of ambient noise levels and coda click source levels with models of sound propagation to estimate the active space of coda communication. Coda clicks were localized off the island of Dominica with a four- or five-element 80 m vertical hydrophone array, allowing us to calculate the median RMS source levels of 1598 clicks from 444 codas to be 161 dB re. 1 µPa (IQR 153-167), placing codas among the most powerful communication sounds in toothed whales. However, together with measured ambient noise levels, these source levels lead to a median active space of coda communication of ∼4 km, reflecting the maximum footprint of a single foraging sperm whale unit. We conclude that while sperm whale codas may contain information about clan affiliation, their moderate active space shows that codas are not used for long range acoustic communication between units and clans, but likely serve to mediate social cohesion and behavioral transitions in intra-unit communication.

Behavioural responses of fin whales to military mid-frequency active sonar.

The effect of active sonars on marine mammal behaviour is a topic of considerable interest and scientific investigation. Some whales, including the largest species (blue whales, Balaenoptera musculus), can be impacted by mid-frequency (1-10 kHz) military sonars. Here we apply complementary experimental methods to provide the first experimentally controlled measurements of behavioural responses to military sonar and similar stimuli for a related endangered species, fin whales (Balaenoptera physalus). Analytical methods include: (i) principal component analysis paired with generalized additive mixed models; (ii) hidden Markov models; and (iii) structured expert elicitation using response severity metrics. These approaches provide complementary perspectives on the nature of potential changes within and across individuals. Behavioural changes were detected in five of 15 whales during controlled exposure experiments using mid-frequency active sonar or pseudorandom noise of similar frequency, duration and source and received level. No changes were detected during six control (no noise) sequences. Overall responses were more limited in occurrence, severity and duration than in blue whales and were less dependent upon contextual aspects of exposure and more contingent upon exposure received level. Quantifying the factors influencing marine mammal responses to sonar is critical in assessing and mitigating future impacts.

Intestinal endothelial cells increase HIV infection and latency in resting and activated CD4 + T cells, particularly affecting CCR6 + CD4 + T cells.

BACKGROUND: With suppressive antiretroviral therapy, HIV infection is well-managed in most patients. However, eradication and cure are still beyond reach due to latent viral reservoirs in CD4 + T cells, particularly in lymphoid tissue environments including the gut associated lymphatic tissues. In HIV patients, there is extensive depletion of T helper cells, particularly T helper 17 cells from the intestinal mucosal area, and the gut is one of the largest viral reservoir sites. Endothelial cells line lymphatic and blood vessels and were found to promote HIV infection and latency in previous studies. In this study, we examined endothelial cells specific to the gut mucosal area-intestinal endothelial cells-for their impact on HIV infection and latency in T helper cells. RESULTS: We found that intestinal endothelial cells dramatically increased productive and latent HIV infection in resting CD4 + T helper cells. In activated CD4 + T cells, endothelial cells enabled the formation of latent infection in addition to the increase of productive infection. Endothelial-cell-mediated HIV infection was more prominent in memory T cells than naïve T cells, and it involved the cytokine IL-6 but did not involve the co-stimulatory molecule CD2. The CCR6 + T helper 17 subpopulation was particularly susceptible to such endothelial-cell-promoted infection. CONCLUSION: Endothelial cells, which are widely present in lymphoid tissues including the intestinal mucosal area and interact regularly with T cells physiologically, significantly increase HIV infection and latent reservoir formation in CD4 + T cells, particularly in CCR6 + T helper 17 cells. Our study highlighted the importance of endothelial cells and the lymphoid tissue environment in HIV pathology and persistence.

Northern bottlenose whales in a pristine environment respond strongly to close and distant navy sonar signals.

Impact assessments for sonar operations typically use received sound levels to predict behavioural disturbance in marine mammals. However, there are indications that cetaceans may learn to associate exposures from distant sound sources with lower perceived risk. To investigate the roles of source distance and received level in an area without frequent sonar activity, we conducted multi-scale controlled exposure experiments ( n = 3) with 12 northern bottlenose whales near Jan Mayen, Norway. Animals were tagged with high-resolution archival tags ( n = 1 per experiment) or medium-resolution satellite tags ( n = 9 in total) and subsequently exposed to sonar. We also deployed bottom-moored recorders to acoustically monitor for whales in the exposed area. Tagged whales initiated avoidance of the sound source over a wide range of distances (0.8-28 km), with responses characteristic of beaked whales. Both onset and intensity of response were better predicted by received sound pressure level (SPL) than by source distance. Avoidance threshold SPLs estimated for each whale ranged from 117-126 dB re 1 µPa, comparable to those of other tagged beaked whales. In this pristine underwater acoustic environment, we found no indication that the source distances tested in our experiments modulated the behavioural effects of sonar, as has been suggested for locations where whales are frequently exposed to sonar.

Behavioral responses of individual blue whales (Balaenoptera musculus) to mid-frequency military sonar.

This study measured the degree of behavioral responses in blue whales (Balaenoptera musculus) to controlled noise exposure off the southern California coast. High-resolution movement and passive acoustic data were obtained from non-invasive archival tags (n=42) whereas surface positions were obtained with visual focal follows. Controlled exposure experiments (CEEs) were used to obtain direct behavioral measurements before, during and after simulated and operational military mid-frequency active sonar (MFAS), pseudorandom noise (PRN) and controls (no noise exposure). For a subset of deep-feeding animals (n=21), active acoustic measurements of prey were obtained and used as contextual covariates in response analyses. To investigate potential behavioral changes within individuals as a function of controlled noise exposure conditions, two parallel analyses of time-series data for selected behavioral parameters (e.g. diving, horizontal movement and feeding) were conducted. This included expert scoring of responses according to a specified behavioral severity rating paradigm and quantitative change-point analyses using Mahalanobis distance statistics. Both methods identified clear changes in some conditions. More than 50% of blue whales in deep-feeding states responded during CEEs, whereas no changes in behavior were identified in shallow-feeding blue whales. Overall, responses were generally brief, of low to moderate severity, and highly dependent on exposure context such as behavioral state, source-to-whale horizontal range and prey availability. Response probability did not follow a simple exposure-response model based on received exposure level. These results, in combination with additional analytical methods to investigate different aspects of potential responses within and among individuals, provide a comprehensive evaluation of how free-ranging blue whales responded to mid-frequency military sonar.

Vocal foragers and silent crowds: context-dependent vocal variation in Northeast Atlantic long-finned pilot whales.

ABSTRACT: Vocalisations form a key component of the social interactions and foraging behaviour of toothed whales. We investigated changes in calling and echolocation behaviour of long-finned pilot whales between foraging and non-foraging periods, by combining acoustic recordings and diving depth data from tagged individuals with concurrent surface observations on social behaviour of their group. The pilot whales showed marked vocal variation, specific to foraging and social context. During periods of foraging, pilot whales showed more vocal activity than during non-foraging periods (rest, travel). In addition to the expected increase in echolocation activity, call rates also increased, suggesting that pilot whales communicate more during foraging. Furthermore, calls with multiple inflections occurred more often immediately before and after foraging dives and during the early descent and late ascent phases of foraging dives. However, these calls were almost never detected at diving depths of the tagged whale beyond 350 m. Calls with no or few inflections were produced at all times, irrespective of diving depth of the tagged whale. We discuss possible explanations for the distinct vocal variation associated with foraging periods. In addition, during non-foraging periods, the pilot whales were found to be more silent (no calling or echolocation) in larger, more closely spaced groups. This indicates that increased levels of social cohesion may release the need to stay in touch acoustically. SIGNIFICANCE STATEMENT: Social toothed whales rely on vocalisations to find prey and interact with conspecifics. Species are often highly vocal and can have elaborate call repertoires. However, it often remains unclear how their repertoire use correlates to specific social and behavioural contexts, which is vital to understand toothed whale foraging strategies and sociality. Combining on-animal tag recordings of diving and acoustic behaviour with observations of social behaviour, we found that pilot whales produce more calls during foraging than during non-foraging periods. Moreover, highly inflected calls were closely associated to the periods around and during foraging dives. This indicates enhanced communication during foraging, which may, for example, enable relocation of conspecifics or sharing of information. Whales reduced their vocal activity (calling and echolocation) at increased levels of social cohesion, indicating that in certain behavioural contexts, closer association (i.e. more closely spaced) may release the need to stay in touch acoustically.

Diving behaviour of Cuvier's beaked whales exposed to two types of military sonar.

Cuvier's beaked whales (Ziphius cavirostris) have stranded in association with mid-frequency active sonar (MFAS) use, and though the causative mechanism linking these events remains unclear, it is believed to be behaviourally mediated. To determine whether MFAS use was associated with behavioural changes in this species, satellite tags were used to record the diving and movements of 16 Cuvier's beaked whales for up to 88 days in a region of frequent MFAS training off the coast of Southern California. Tag data were combined with summarized records of concurrent bouts of high-power, surface-ship and mid-power, helicopter-deployed MFAS use, along with other potential covariates, in generalized additive mixed-effects models. Deep dives, shallow dives and surface intervals tended to become longer during MFAS use, with some variation associated with the total amount of overlapping MFAS during the behaviour. These changes in dives and surface intervals contributed to a longer interval between deep dives, a proxy for foraging disruption in this species. Most responses intensified with proximity and were more pronounced during mid-power than high-power MFAS use at comparable distances within approximately 50 km, despite the significantly lower source level of mid-power MFAS. However, distance-mediated responses to high-power MFAS, and increased deep dive intervals during mid-power MFAS, were evident up to approximately 100 km away.

An analysis of pilot whale vocalization activity using hidden Markov models.

Vocalizations of cetaceans form a key component of their social interactions. Such vocalization activity is driven by the behavioral states of the whales, which are not directly observable, so that latent-state models are natural candidates for modeling empirical data on vocalizations. In this paper, hidden Markov models are used to analyze calling activity of long-finned pilot whales (Globicephala melas) recorded over three years in the Vestfjord basin off Lofoten, Norway. Baseline models are used to motivate the use of three states, while more complex models are fit to study the influence of covariates on the state-switching dynamics. The analysis demonstrates the potential usefulness of hidden Markov models to concisely yet accurately describe the stochastic patterns found in animal communication data, thereby providing a framework for drawing meaningful biological inference.

The Challenges of Analyzing Behavioral Response Study Data: An Overview of the MOCHA (Multi-study OCean Acoustics Human Effects Analysis) Project.

This paper describes the MOCHA project which aims to develop novel approaches for the analysis of data collected during Behavioral Response Studies (BRSs). BRSs are experiments aimed at directly quantifying the effects of controlled dosages of natural or anthropogenic stimuli (typically sound) on marine mammal behavior. These experiments typically result in low sample size, relative to variability, and so we are looking at a number of studies in combination to maximize the gain from each one. We describe a suite of analytical tools applied to BRS data on beaked whales, including a simulation study aimed at informing future experimental design.

Acoustic sequences in non-human animals: a tutorial review and prospectus.

Animal acoustic communication often takes the form of complex sequences, made up of multiple distinct acoustic units. Apart from the well-known example of birdsong, other animals such as insects, amphibians, and mammals (including bats, rodents, primates, and cetaceans) also generate complex acoustic sequences. Occasionally, such as with birdsong, the adaptive role of these sequences seems clear (e.g. mate attraction and territorial defence). More often however, researchers have only begun to characterise - let alone understand - the significance and meaning of acoustic sequences. Hypotheses abound, but there is little agreement as to how sequences should be defined and analysed. Our review aims to outline suitable methods for testing these hypotheses, and to describe the major limitations to our current and near-future knowledge on questions of acoustic sequences. This review and prospectus is the result of a collaborative effort between 43 scientists from the fields of animal behaviour, ecology and evolution, signal processing, machine learning, quantitative linguistics, and information theory, who gathered for a 2013 workshop entitled, 'Analysing vocal sequences in animals'. Our goal is to present not just a review of the state of the art, but to propose a methodological framework that summarises what we suggest are the best practices for research in this field, across taxa and across disciplines. We also provide a tutorial-style introduction to some of the most promising algorithmic approaches for analysing sequences. We divide our review into three sections: identifying the distinct units of an acoustic sequence, describing the different ways that information can be contained within a sequence, and analysing the structure of that sequence. Each of these sections is further subdivided to address the key questions and approaches in that area. We propose a uniform, systematic, and comprehensive approach to studying sequences, with the goal of clarifying research terms used in different fields, and facilitating collaboration and comparative studies. Allowing greater interdisciplinary collaboration will facilitate the investigation of many important questions in the evolution of communication and sociality.

Nonparametric inference in hidden Markov models using P-splines.

Hidden Markov models (HMMs) are flexible time series models in which the distribution of the observations depends on unobserved serially correlated states. The state-dependent distributions in HMMs are usually taken from some class of parametrically specified distributions. The choice of this class can be difficult, and an unfortunate choice can have serious consequences for example on state estimates, and more generally on the resulting model complexity and interpretation. We demonstrate these practical issues in a real data application concerned with vertical speeds of a diving beaked whale, where we demonstrate that parametric approaches can easily lead to overly complex state processes, impeding meaningful biological inference. In contrast, for the dive data, HMMs with nonparametrically estimated state-dependent distributions are much more parsimonious in terms of the number of states and easier to interpret, while fitting the data equally well. Our nonparametric estimation approach is based on the idea of representing the densities of the state-dependent distributions as linear combinations of a large number of standardized B-spline basis functions, imposing a penalty term on non-smoothness in order to maintain a good balance between goodness-of-fit and smoothness.

First direct measurements of behavioural responses by Cuvier's beaked whales to mid-frequency active sonar.

Most marine mammal- strandings coincident with naval sonar exercises have involved Cuvier's beaked whales (Ziphius cavirostris). We recorded animal movement and acoustic data on two tagged Ziphius and obtained the first direct measurements of behavioural responses of this species to mid-frequency active (MFA) sonar signals. Each recording included a 30-min playback (one 1.6-s simulated MFA sonar signal repeated every 25 s); one whale was also incidentally exposed to MFA sonar from distant naval exercises. Whales responded strongly to playbacks at low received levels (RLs; 89-127 dB re 1 µPa): after ceasing normal fluking and echolocation, they swam rapidly, silently away, extending both dive duration and subsequent non-foraging interval. Distant sonar exercises (78-106 dB re 1 µPa) did not elicit such responses, suggesting that context may moderate reactions. The observed responses to playback occurred at RLs well below current regulatory thresholds; equivalent responses to operational sonars could elevate stranding risk and reduce foraging efficiency.

Blue whales respond to simulated mid-frequency military sonar.

Mid-frequency military (1-10 kHz) sonars have been associated with lethal mass strandings of deep-diving toothed whales, but the effects on endangered baleen whale species are virtually unknown. Here, we used controlled exposure experiments with simulated military sonar and other mid-frequency sounds to measure behavioural responses of tagged blue whales (Balaenoptera musculus) in feeding areas within the Southern California Bight. Despite using source levels orders of magnitude below some operational military systems, our results demonstrate that mid-frequency sound can significantly affect blue whale behaviour, especially during deep feeding modes. When a response occurred, behavioural changes varied widely from cessation of deep feeding to increased swimming speed and directed travel away from the sound source. The variability of these behavioural responses was largely influenced by a complex interaction of behavioural state, the type of mid-frequency sound and received sound level. Sonar-induced disruption of feeding and displacement from high-quality prey patches could have significant and previously undocumented impacts on baleen whale foraging ecology, individual fitness and population health.

Underwater acrobatics by the world's largest predator: 360° rolling manoeuvres by lunge-feeding blue whales.

The extreme body size of blue whales requires a high energy intake and therefore demands efficient foraging strategies. As an obligate lunge feeder on aggregations of small zooplankton, blue whales engulf a large volume of prey-laden water in a single, rapid gulp. The efficiency of this feeding mechanism is strongly dependent on the amount of prey that can be captured during each lunge, yet food resources tend to be patchily distributed in both space and time. Here, we measured the three-dimensional kinematics and foraging behaviour of blue whales feeding on krill, using suction-cup attached multi-sensor tags. Our analyses revealed 360° rolling lunge-feeding manoeuvres that reorient the body and position the lower jaws so that a krill patch can be engulfed with the whale's body inverted. We also recorded these rolling behaviours when whales were in a searching mode in between lunges, suggesting that this behaviour also enables the whale to visually process the prey field and maximize foraging efficiency by surveying for the densest prey aggregations. These results reveal the complex manoeuvrability that is required for large rorqual whales to exploit prey patches and highlight the need to fully understand the three-dimensional interactions between predator and prey in the natural environment.

Propagation of narrow-band-high-frequency clicks: measured and modeled transmission loss of porpoise-like clicks in porpoise habitats.

Estimating the range at which harbor porpoises can detect prey items and environmental objects is integral to understanding their biosonar. Understanding the ranges at which they can use echolocation to detect and avoid obstacles is particularly important for strategies to reduce bycatch. Transmission loss (TL) during acoustic propagation is an important determinant of those detection ranges, and it also influences animal detection functions used in passive acoustic monitoring. However, common assumptions regarding TL have rarely been tested. Here, TL of synthetic porpoise clicks was measured in porpoise habitats in Canada and Denmark, and field data were compared with spherical spreading law and ray-trace (Bellhop) model predictions. Both models matched mean observations quite well in most cases, indicating that a spherical spreading law can usually provide an accurate first-order estimate of TL for porpoise sounds in porpoise habitat. However, TL varied significantly (+/-10 dB) between sites and over time in response to variability in seafloor characteristics, sound-speed profiles, and other short-timescale environmental fluctuations. Such variability should be taken into account in estimates of the ranges at which porpoises can communicate acoustically, detect echolocation targets, and be detected via passive acoustic monitoring.

Acoustic behaviour of echolocating porpoises during prey capture.

Porpoise echolocation has been studied previously, mainly in target detection experiments using stationed animals and steel sphere targets, but little is known about the acoustic behaviour of free-swimming porpoises echolocating for prey. Here, we used small onboard sound and orientation recording tags to study the echolocation behaviour of free-swimming trained porpoises as they caught dead, freely drifting fish. We analysed porpoise echolocation behaviour leading up to and following prey capture events, including variability in echolocation in response to vision restriction, prey species, and individual porpoise tested. The porpoises produced echolocation clicks as they searched for the fish, followed by fast-repetition-rate clicks (echolocation buzzes) when acquiring prey. During buzzes, which usually began when porpoises were about 1-2 body lengths from prey, tag-recorded click levels decreased by about 10 dB, click rates increased to over 300 clicks per second, and variability in body orientation (roll) increased. Buzzes generally continued beyond the first contact with the fish, and often extended until or after the end of prey handling. This unexplained continuation of buzzes after prey capture raises questions about the function of buzzes, suggesting that in addition to providing detailed information on target location during the capture, they may serve additional purposes such as the relocation of potentially escaping prey. We conclude that porpoises display the same overall acoustic prey capture behaviour seen in larger toothed whales in the wild, albeit at a faster pace, clicking slowly during search and approach phases and buzzing during prey capture.

Modeling acoustic propagation of airgun array pulses recorded on tagged sperm whales (Physeter macrocephalus).

In 2002 and 2003, tagged sperm whales (Physeter macrocephalus) were experimentally exposed to airgun pulses in the Gulf of Mexico, with the tags providing acoustic recordings at measured ranges and depths. Ray trace and parabolic equation (PE) models provided information about sound propagation paths and accurately predicted time of arrival differences between multipath arrivals. With adequate environmental information, a broadband acoustic PE model predicted the relative levels of multipath arrivals recorded on the tagged whales. However, lack of array source signature data limited modeling of absolute received levels. Airguns produce energy primarily below 250 Hz, with spectrum levels about 20-40 dB lower at 1 kHz. Some arrivals recorded near the surface in 2002 had energy predominantly above 500 Hz; a surface duct in the 2002 sound speed profile helps explain this effect, and the beampattern of the source array also indicates an increased proportion of high-frequency sound at near-horizontal launch angles. These findings indicate that airguns sometimes expose animals to measurable sound energy above 250 Hz, and demonstrate the influences of source and environmental parameters on characteristics of received airgun pulses. The study also illustrates that on-axis source levels and simple geometric spreading inadequately describe airgun pulse propagation and the extent of exposure zones.

Akt regulates basic helix-loop-helix transcription factor-coactivator complex formation and activity during neuronal differentiation.

Neural basic helix-loop-helix (bHLH) transcription factors regulate neurogenesis in vertebrates. Signaling by peptide growth factors also plays critical roles in regulating neuronal differentiation and survival. Many peptide growth factors activate phosphatidylinositol 3-kinase (PI3K) and subsequently the Akt kinases, raising the possibility that Akt may impact bHLH protein function during neurogenesis. Here we demonstrate that reducing expression of endogenous Akt1 and Akt2 by RNA interference (RNAi) reduces neuron generation in P19 cells transfected with a neural bHLH expression vector. The reduction in neuron generation from decreased Akt expression is not solely due to decreased cell survival, since addition of the caspase inhibitor z-VAD-FMK rescues cell death associated with loss of Akt function but does not restore neuron formation. This result indicates that Akt1 and Akt2 have additional functions during neuronal differentiation that are separable from neuronal survival. We show that activated Akt1 enhances complex formation between bHLH proteins and the transcriptional coactivator p300. Activated Akt1 also significantly augments the transcriptional activity of the bHLH protein neurogenin 3 in complex with the coactivators p300 or CBP. In addition, inhibition of endogenous Akt activity by the PI3K/Akt inhibitor LY294002 abolishes transcriptional cooperativity between the bHLH proteins and p300. We propose that Akt regulates the assembly and activity of bHLH-coactivator complexes to promote neuronal differentiation.

Simultaneous inhibition of GSK3alpha and GSK3beta using hairpin siRNA expression vectors.

Short interfering RNAs (siRNAs) can mediate sequence-specific inhibition of gene expression in mammalian cells. We and others have recently developed expression vector-based systems for synthesizing siRNAs or hairpin siRNAs in mammalian cells. Expression vector-based RNA interference (RNAi) effectively suppresses expression of target genes and is likely to be a powerful tool for analysis of gene function. Here we compare inhibition by vectors expressing hairpin siRNA designs either with different loop sequences connecting the two siRNA strands, or with duplex regions of different lengths. Our results suggest that lengthening the 19-nucleotide duplex region of a relatively ineffective hairpin siRNA can increase inhibition, but increasing the length of an effective 19-nt hairpin siRNA does not increase inhibition. We also demonstrate that hairpin siRNA vectors can be used to inhibit two target genes simultaneously. We have targeted glycogen synthase kinase-3alpha (GSK-3alpha) and GSK-3beta, two related kinases involved in the regulation of a variety of cellular processes and also implicated in the pathogenesis of several human diseases. Inhibition of either GSK-3alpha or GSK-3beta by transfection of hairpin siRNA vectors leads to elevated expression of the GSK-3 target beta-catenin, whereas inhibition of both kinases further increases beta-catenin expression. Our results suggest that vector-based siRNA inhibition may be useful for dissecting the functional roles of GSK-3alpha and GSK-3beta in somatic cells. The ability to inhibit two or more genes simultaneously with hairpin siRNA expression vectors should facilitate studies of gene function in mammalian cells.

RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells.

Duplexes of 21-nt RNAs, known as short-interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference (RNAi) when introduced into mammalian cells. We show that siRNAs can be synthesized by in vitro transcription with T7 RNA polymerase, providing an economical alternative to chemical synthesis of siRNAs. By using this method, we show that short hairpin siRNAs can function like siRNA duplexes to inhibit gene expression in a sequence-specific manner. Further, we find that hairpin siRNAs or siRNAs expressed from an RNA polymerase III vector based on the mouse U6 RNA promoter can effectively inhibit gene expression in mammalian cells. U6-driven hairpin siRNAs dramatically reduced the expression of a neuron-specific beta-tubulin protein during the neuronal differentiation of mouse P19 cells, demonstrating that this approach should be useful for studies of differentiation and neurogenesis. We also observe that mismatches within hairpin siRNAs can increase the strand selectivity of a hairpin siRNA, which may reduce self-targeting of vectors expressing siRNAs. Use of hairpin siRNA expression vectors for RNAi should provide a rapid and versatile method for assessing gene function in mammalian cells, and may have applications in gene therapy.