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 ␤-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. RNA interference (RNAi) has become a powerful and widely used tool for the analysis of gene function in invertebrates and plants (reviewed in ref. 1). Introduction of double-stranded RNA (dsRNA) into the cells of these organisms leads to the sequence-specific destruction of endogenous RNAs that match the dsRNA. During RNAi, long dsRNA molecules are processed into 19-to 23-nt RNAs known as short-interfering RNAs (siRNAs) that serve as guides for enzymatic cleavage of complementary RNAs (2-10). In addition, siRNAs can function as primers for an RNA-dependent RNA polymerase that synthesizes additional dsRNA, which in turn is processed into siRNAs, amplifying the effects of the original siRNAs (11,12). Although the overall process of siRNA inhibition has been characterized, the specific enzymes that mediate siRNA function remain to be identified.In mammalian cells, dsRNA is processed into siRNAs (13-16), but RNAi with dsRNA has not been successful in most cell types because of nonspecific responses elicited by dsRNA molecules longer than about 30 nt (17). However, Tuschl and coworkers (13, 18) recently made the remarkable observation that transfection of synthetic 21-nt siRNA duplexes into mammalian cells effectively inhibits endogenous genes in a sequence-specific manner. These siRNA duplexes are too short to trigger the nonspecific dsRNA responses, but they still cause destruction of complementary RNA sequences (19). It is not known whether siRNAs in mammalian cells also prime synthesis of dsRNA to form additional siRNAs. The recent discovery of large numbers of microRNA genes (reviewed in ref. 20) raises the prospect that the cellular machinery necessary for siRNA inhibition in mammalian cells may be linked to normal processes of gene regulation.In the hope of applying...

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.

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.

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.

Characterization of multivariate time series of behaviour data from animal-borne sensors is challenging. Biologists require methods to objectively quantify baseline behaviour, then assess behaviour changes in response to environmental stimuli. Here, we apply hidden Markov models (HMMs) to characterize blue whale movement and diving behaviour, identifying latent states corresponding to three main underlying behaviour states: shallow feeding, travelling, and deep feeding. The model formulation accounts for inter-whale differences via a computationally efficient discrete random effect, and measures potential effects of experimental acoustic disturbance on between-state transition probabilities. We identify clear differences in blue whale disturbance response depending on the behavioural context during exposure, with whales less likely to initiate deep foraging behaviour during exposure. Findings are consistent with earlier studies using smaller samples, but the HMM approach provides more nuanced characterization of behaviour changes.

The largest animals are marine filter feeders, but the underlying mechanism of their large size remains unexplained. We measured feeding performance and prey quality to demonstrate how whale gigantism is driven by the interplay of prey abundance and harvesting mechanisms that increase prey capture rates and energy intake. The foraging efficiency of toothed whales that feed on single prey is constrained by the abundance of large prey, whereas filter-feeding baleen whales seasonally exploit vast swarms of small prey at high efficiencies. Given temporally and spatially aggregated prey, filter feeding provides an evolutionary pathway to extremes in body size that are not available to lineages that must feed on one prey at a time. Maximum size in filter feeders is likely constrained by prey availability across space and time.

SUMMARYPorpoise 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.

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