Expression of Mn-sod, PAL1, aos1 and HPL genes in soybean plants overexpressing the NmDef02 defensin.

Plant defensins are a potential tool in crop improvement programs through biotechnology. Their antifungal action makes them attractive molecules for the production of transgenic plants. Information is currently lacking on what happens to the expression of defense genes in transgenic plants that overexpress a defensin. Here we show the relative expression of four defense-related genes: Mn-sod, PAL1, aos1 and HPL evaluated in two transgenic soybean events (Def1 and Def17) constitutively expressing the NmDef02 defensin gene from Nicotiana megalosiphon. The expression of these defense genes showed a differential profile in the transgenic events, with the increased expression of the aos1 gene and the repression of the Mn-sod gene in both events, when compared to the non-transgenic control. Furthermore, the expression of the PAL1 gene only increased in the Def17 event. The results indicate that although there were some changes in the expression of defense genes in transgenic plants overexpressing the defensin NmDef02; the morphoagronomic parameters evaluated were similar to the non-transgenic control. Understanding the molecular changes that occur in these transgenic plants could be of interest in the short, medium and long term.

Echolocating toothed whales use ultra-fast echo-kinetic responses to track evasive prey.

Visual predators rely on fast-acting optokinetic responses to track and capture agile prey. Most toothed whales, however, rely on echolocation for hunting and have converged on biosonar clicking rates reaching 500/s during prey pursuits. If echoes are processed on a click-by-click basis, as assumed, neural responses 100× faster than those in vision are required to keep pace with this information flow. Using high-resolution biologging of wild predator-prey interactions, we show that toothed whales adjust clicking rates to track prey movement within 50-200 ms of prey escape responses. Hypothesising that these stereotyped biosonar adjustments are elicited by sudden prey accelerations, we measured echo-kinetic responses from trained harbour porpoises to a moving target and found similar latencies. High biosonar sampling rates are, therefore, not supported by extreme speeds of neural processing and muscular responses. Instead, the neurokinetic response times in echolocation are similar to those of tracking responses in vision, suggesting a common neural underpinning.

In the animal world, split-second decisions determine whether a predator eats, or its prey survives. There is a strong evolutionary advantage to fast reacting brains and bodies. For example, the eye muscles of hunting cheetahs must lock on to a gazelle and keep track of it, no matter how quickly or unpredictably it moves. In fact, in monkeys and primates, these muscles can react to sudden movements in as little as 50 milliseconds ­ faster than the blink of an eye. But what about animals that do not rely on vision to hunt? To find food at night or in the deep ocean, whales and porpoises make short ultrasonic sounds, or 'clicks', and then listen for returning echoes. As they close in on a prey, they need to click faster to get quicker updates on its location. What is unclear is how fast they react to the echoes. Just before a kill, a harbour porpoise can click over 500 times a second: if they wait for the echo from one click before making the next one, they would need responses 100 times faster than human eyes. Exploring this topic is difficult, as it requires tracking predator and prey at the same time. Vance et al. took up the challenge by building sound and movement recorders that attach to whales with suction cups. These were used on two different hunters: deep-diving beaked whales and shallow-hunting harbour porpoises. Both species adapted their click rate depending on how far they were from their prey, but their response times were similar to visual responses in monkeys and humans. This means that whales and porpoises do not act on each echo before clicking again: instead, they respond to groups of tens of clicks at a time. This suggests that their brains may be wired in much the same way as the ones of visual animals. In the ocean, increased human activity creates a dangerous noise pollution that disrupts the delicate hunting mechanism of whales and porpoises. Better understanding how these animals find their food may therefore help conservation efforts.

Large-scale movements of common bottlenose dolphins in the Atlantic: dolphins with an international courtyard.

Wide-ranging connectivity patterns of common bottlenose dolphins (Tursiops truncatus) are generally poorly known worldwide and more so within the oceanic archipelagos of Macaronesia in the North East (NE) Atlantic. This study aimed to identify long-range movements between the archipelagos of Macaronesia that lie between 500 and 1,500 km apart, and between Madeira archipelago and the Portuguese continental shelf, through the compilation and comparison of bottlenose dolphin's photo-identification catalogues from different regions: one from Madeira (n = 363 individuals), two from different areas in the Azores (n = 495 and 176), and four from different islands of the Canary Islands (n = 182, 110, 142 and 281), summing up 1791 photographs. An additional comparison was made between the Madeira catalogue and one catalogue from Sagres, on the southwest tip of the Iberian Peninsula (n = 359). Results showed 26 individual matches, mostly between Madeira and the Canary Islands (n = 23), and between Azores and Madeira (n = 3). No matches were found between the Canary Islands and the Azores, nor between Madeira and Sagres. There were no individuals identified in all three archipelagos. The minimum time recorded between sightings in two different archipelagos (≈ 460 km apart) was 62 days. Association patterns revealed that the individuals moving between archipelagos were connected to resident, migrant and transient individuals in Madeira. The higher number of individuals that were re-sighted between Madeira and the Canary Islands can be explained by the relative proximity of these two archipelagos. This study shows the first inter-archipelago movements of bottlenose dolphins in the Macaronesia region, emphasizing the high mobility of this species and supporting the high gene flow described for oceanic dolphins inhabiting the North Atlantic. The dynamics of these long-range movements strongly denotes the need to review marine protected areas established for this species in each archipelago, calling for joint resolutions from three autonomous regions belonging to two EU countries.

Deep-diving beaked whales dive together but forage apart.

Echolocating animals that forage in social groups can potentially benefit from eavesdropping on other group members, cooperative foraging or social defence, but may also face problems of acoustic interference and intra-group competition for prey. Here, we investigate these potential trade-offs of sociality for extreme deep-diving Blainville's and Cuvier's beaked whales. These species perform highly synchronous group dives as a presumed predator-avoidance behaviour, but the benefits and costs of this on foraging have not been investigated. We show that group members could hear their companions for a median of at least 91% of the vocal foraging phase of their dives. This enables whales to coordinate their mean travel direction despite differing individual headings as they pursue prey on a minute-by-minute basis. While beaked whales coordinate their echolocation-based foraging periods tightly, individual click and buzz rates are both independent of the number of whales in the group. Thus, their foraging performance is not affected by intra-group competition or interference from group members, and they do not seem to capitalize directly on eavesdropping on the echoes produced by the echolocation clicks of their companions. We conclude that the close diving and vocal synchronization of beaked whale groups that quantitatively reduces predation risk has little impact on foraging performance.

Field Resistance to Phakopsora pachyrhizi and Colletotrichum truncatum of Transgenic Soybean Expressing the NmDef02 Plant Defensin Gene.

Fungal diseases lead to significant losses in soybean yields and a decline in seed quality; such is the case of the Asian soybean rust and anthracnose caused by Phakopsora pachyrhizi and Colletotrichum truncatum, respectively. Currently, the development of transgenic plants carrying antifungal defensins offers an alternative for plant protection against pathogens. This paper shows the production of transgenic soybean plants expressing the NmDef02 defensin gene using the biolistic delivery system, in an attempt to improve resistance against diseases and reduce the need for chemicals. Transgenic lines were assessed in field conditions under the natural infections of P. pachyrhizi and C. truncatum. The constitutive expression of the NmDef02 gene in transgenic soybean plants was shown to enhance resistance against these important plant pathogens. The quantification of the P. pachyrhizi biomass in infected soybean leaves revealed significant differences between transgenic lines and the non-transgenic control. In certain transgenic lines there was a strong reduction of fungal biomass, revealing a less severe disease. Integration and expression of the transgenes were confirmed by PCR, Southern blot, and qRT-PCR, where the Def1 line showed a higher relative expression of defensin. It was also found that the expression of the NmDef02 defensin gene in plants of the Def1 line did not have a negative effect on the nodulation induced by Bradyrhizobium japonicum. These results indicate that transgenic soybean plants expressing the NmDef02 defensin gene have a substantially enhanced resistance to economically important diseases, providing a sound environmental approach for decreasing yield losses and lowering the burden of chemicals in agriculture.

Fear of Killer Whales Drives Extreme Synchrony in Deep Diving Beaked Whales.

Fear of predation can induce profound changes in the behaviour and physiology of prey species even if predator encounters are infrequent. For echolocating toothed whales, the use of sound to forage exposes them to detection by eavesdropping predators, but while some species exploit social defences or produce cryptic acoustic signals, deep-diving beaked whales, well known for mass-strandings induced by navy sonar, seem enigmatically defenceless against their main predator, killer whales. Here we test the hypothesis that the stereotyped group diving and vocal behaviour of beaked whales has benefits for abatement of predation risk and thus could have been driven by fear of predation over evolutionary time. Biologging data from 14 Blainville's and 12 Cuvier's beaked whales show that group members have an extreme synchronicity, overlapping vocal foraging time by 98% despite hunting individually, thereby reducing group temporal availability for acoustic detection by killer whales to <25%. Groups also perform a coordinated silent ascent in an unpredictable direction, covering a mean of 1 km horizontal distance from their last vocal position. This tactic sacrifices 35% of foraging time but reduces by an order of magnitude the risk of interception by killer whales. These predator abatement behaviours have likely served beaked whales over millions of years, but may become maladaptive by playing a role in mass strandings induced by man-made predator-like sonar sounds.

Deep-diving pilot whales make cheap, but powerful, echolocation clicks with 50 µL of air.

Echolocating toothed whales produce powerful clicks pneumatically to detect prey in the deep sea where this long-range sensory channel makes them formidable top predators. However, air supplies for sound production compress with depth following Boyle's law suggesting that deep-diving whales must use very small air volumes per echolocation click to facilitate continuous sensory flow in foraging dives. Here we test this hypothesis by analysing click-induced acoustic resonances in the nasal air sacs, recorded by biologging tags. Using 27000 clicks from 102 dives of 23 tagged pilot whales (Globicephala macrorhynchus), we show that click production requires only 50 µL of air/click at 500 m depth increasing gradually to 100 µL at 1000 m. With such small air volumes, the metabolic cost of sound production is on the order of 40 J per dive which is a negligible fraction of the field metabolic rate. Nonetheless, whales must make frequent pauses in echolocation to recycle air between nasal sacs. Thus, frugal use of air and periodic recycling of very limited air volumes enable pilot whales, and likely other toothed whales, to echolocate cheaply and almost continuously throughout foraging dives, providing them with a strong sensory advantage in diverse aquatic habitats.

Spatio-temporal variation in click production rates of beaked whales: Implications for passive acoustic density estimation.

Passive acoustic monitoring has become an increasingly prevalent tool for estimating density of marine mammals, such as beaked whales, which vocalize often but are difficult to survey visually. Counts of acoustic cues (e.g., vocalizations), when corrected for detection probability, can be translated into animal density estimates by applying an individual cue production rate multiplier. It is essential to understand variation in these rates to avoid biased estimates. The most direct way to measure cue production rate is with animal-mounted acoustic recorders. This study utilized data from sound recording tags deployed on Blainville's (Mesoplodon densirostris, 19 deployments) and Cuvier's (Ziphius cavirostris, 16 deployments) beaked whales, in two locations per species, to explore spatial and temporal variation in click production rates. No spatial or temporal variation was detected within the average click production rate of Blainville's beaked whales when calculated over dive cycles (including silent periods between dives); however, spatial variation was detected when averaged only over vocal periods. Cuvier's beaked whales exhibited significant spatial and temporal variation in click production rates within vocal periods and when silent periods were included. This evidence of variation emphasizes the need to utilize appropriate cue production rates when estimating density from passive acoustic data.

True's beaked whale (Mesoplodon mirus) in Macaronesia.

The True's beaked whale (Mesoplodon mirus, True 1913) is a poorly known member of the Ziphiidae family. Its distribution in the northern hemisphere is thought to be restricted to the temperate or warm temperate waters of the North Atlantic, while a few stranding records from the southern hemisphere suggest a wider and antitropical distribution, extending to waters from the Atlantic coast of Brazil to South Africa, Mozambique, Australia and the Tasman Sea coast of New Zealand. This paper (i) reports the first molecular confirmation of the occurrence of the True's beaked whale at the southern limit of its distribution recorded in the northeast Atlantic: the Azores and Canary Islands (macaronesian ecoregion); (ii) describes a new colouration for this species using evidence from a whale with molecular species confirmation; and (iii) contributes to the sparse worldwide database of live sightings, including the first underwater video recording of this species and close images of a calf. Species identification was confirmed in two cases using mitochondrial DNA control region and cytochrome b gene markers: a subadult male True's beaked whale that stranded in El Hierro, Canary Islands, in November 2012, and a subadult male found floating dead near Faial, the Azores, in July 2004. The whale that stranded in the Canary Islands had a clearly delimited white area on its head, extending posteriorly from the tip of the beak to cover the blowhole dorsally and the gular grooves ventrally. This colouration contrasts with previous descriptions for the species and it may be rare, but it exemplifies the variability of the colouration of True's beaked whales in the North Atlantic, further confirmed here by live sightings data. The recording of several observations of this species in deep but relatively coastal waters off the Azores and the Canary Islands suggests that these archipelagos may be unique locations to study the behaviour of the enigmatic True's beaked whale.

Correction: Abundance and Distribution of Sperm Whales in the Canary Islands: Can Sperm Whales in the Archipelago Sustain the Current Level of Ship-Strike Mortalities?

[This corrects the article DOI: 10.1371/journal.pone.0150660.].

Tracking the kinematics of caudal-oscillatory swimming: a comparison of two on-animal sensing methods.

Studies of locomotion kinematics require high-resolution information about body movements and the specific acceleration (SA) that these generate. On-animal accelerometers measure both orientation and SA but an additional orientation sensor is needed to accurately separate these. Although gyroscopes can perform this function, their power consumption, drift and complex data processing make them unattractive for biologging. Lower power magnetometers can also be used with some limitations. Here, we present an integrated and simplified method for estimating body rotations and SA applicable to both gyroscopes and magnetometers, enabling a direct comparison of these two sensors. We use a tag with both sensors to demonstrate how caudal-oscillation rate and SA are adjusted by a diving whale in response to rapidly changing buoyancy forces as the lungs compress while descending. The two sensors gave similar estimates of the dynamic forces, demonstrating that magnetometers may offer a simpler low-power alternative for miniature tags in some applications.

Abundance and Distribution of Sperm Whales in the Canary Islands: Can Sperm Whales in the Archipelago Sustain the Current Level of Ship-Strike Mortalities?

Sperm whales are present in the Canary Islands year-round, suggesting that the archipelago is an important area for this species in the North Atlantic. However, the area experiences one of the highest reported rates of sperm whale ship-strike in the world. Here we investigate if the number of sperm whales found in the archipelago can sustain the current rate of ship-strike mortality. The results of this study may also have implications for offshore areas where concentrations of sperm whales may coincide with high densities of ship traffic, but where ship-strikes may be undocumented. The absolute abundance of sperm whales in an area of 52933 km2, covering the territorial waters of the Canary Islands, was estimated from 2668 km of acoustic line-transect survey using Distance sampling analysis. Data on sperm whale diving and acoustic behaviour, obtained from bio-logging, were used to calculate g(0) = 0.92, this is less than one because of occasional extended periods when whales do not echolocate. This resulted in an absolute abundance estimate of 224 sperm whales (95% log-normal CI 120-418) within the survey area. The recruitment capability of this number of whales, some 2.5 whales per year, is likely to be exceeded by the current ship-strike mortality rate. Furthermore, we found areas of higher whale density within the archipelago, many coincident with those previously described, suggesting that these are important habitats for females and immature animals inhabiting the archipelago. Some of these areas are crossed by active shipping lanes increasing the risk of ship-strikes. Given the philopatry in female sperm whales, replacement of impacted whales might be limited. Therefore, the application of mitigation measures to reduce the ship-strike mortality rate seems essential for the conservation of sperm whales in the Canary Islands.

Peer-Reviewed Studies on the Effects of Anthropogenic Noise on Marine Invertebrates: From Scallop Larvae to Giant Squid.

Marine invertebrates at the base of oceanic trophic webs play important ecological and economical roles supporting worldwide fisheries worth millions. There is an increasing concern about the effects of anthropogenic noise on marine fauna but little is known about its effects on invertebrates. Here the current peer-reviewed literature on this subject is reviewed, dealing with different ontogenetic stages and taxa. These studies show that the noise effects on marine invertebrates range from apparently null to behavioral/physiological responses to mortalities. They emphasize the need to consider potential interactions of human activities using intense sound sources with the conservation and fisheries of local invertebrate stocks.

Gait switches in deep-diving beaked whales: biomechanical strategies for long-duration dives.

Diving animals modulate their swimming gaits to promote locomotor efficiency and so enable longer, more productive dives. Beaked whales perform extremely long and deep foraging dives that probably exceed aerobic capacities for some species. Here, we use biomechanical data from suction-cup tags attached to three species of beaked whales (Mesoplodon densirostris, N=10; Ziphius cavirostris, N=9; and Hyperoodon ampullatus, N=2) to characterize their swimming gaits. In addition to continuous stroking and stroke-and-glide gaits described for other diving mammals, all whales produced occasional fluke-strokes with distinctly larger dorso-ventral acceleration, which we termed 'type-B' strokes. These high-power strokes occurred almost exclusively during deep dive ascents as part of a novel mixed gait. To quantify body rotations and specific acceleration generated during strokes we adapted a kinematic method combining data from two sensors in the tag. Body rotations estimated with high-rate magnetometer data were subtracted from accelerometer data to estimate the resulting surge and heave accelerations. Using this method, we show that stroke duration, rotation angle and acceleration were bi-modal for these species, with B-strokes having 76% of the duration, 52% larger body rotation and four times more surge than normal strokes. The additional acceleration of B-strokes did not lead to faster ascents, but rather enabled brief glides, which may improve the overall efficiency of this gait. Their occurrence towards the end of long dives leads us to propose that B-strokes may recruit fast-twitch fibres that comprise ∼80% of swimming muscles in Blainville's beaked whales, thus prolonging foraging time at depth.

Single-click beam patterns suggest dynamic changes to the field of view of echolocating Atlantic spotted dolphins (Stenella frontalis) in the wild.

Echolocating animals exercise an extensive control over the spectral and temporal properties of their biosonar signals to facilitate perception of their actively generated auditory scene when homing in on prey. The intensity and directionality of the biosonar beam defines the field of view of echolocating animals by affecting the acoustic detection range and angular coverage. However, the spatial relationship between an echolocating predator and its prey changes rapidly, resulting in different biosonar requirements throughout prey pursuit and capture. Here, we measured single-click beam patterns using a parametric fit procedure to test whether free-ranging Atlantic spotted dolphins (Stenella frontalis) modify their biosonar beam width. We recorded echolocation clicks using a linear array of receivers and estimated the beam width of individual clicks using a parametric spectral fit, cross-validated with well-established composite beam pattern estimates. The dolphins apparently increased the biosonar beam width, to a large degree without changing the signal frequency, when they approached the recording array. This is comparable to bats that also expand their field of view during prey capture, but achieve this by decreasing biosonar frequency. This behaviour may serve to decrease the risk that rapid escape movements of prey take them outside the biosonar beam of the predator. It is likely that shared sensory requirements have resulted in bats and toothed whales expanding their acoustic field of view at close range to increase the likelihood of successfully acquiring prey using echolocation, representing a case of convergent evolution of echolocation behaviour between these two taxa.

Anthropogenic noise causes body malformations and delays development in marine larvae.

Understanding the impact of noise on marine fauna at the population level requires knowledge about the vulnerability of different life-stages. Here we provide the first evidence that noise exposure during larval development produces body malformations in marine invertebrates. Scallop larvae exposed to playbacks of seismic pulses showed significant developmental delays and 46% developed body abnormalities. Similar effects were observed in all independent samples exposed to noise while no malformations were found in the control groups (4881 larvae examined). Malformations appeared in the D-veliger larval phase, perhaps due to the cumulative exposure attained by this stage or to a greater vulnerability of D-veliger to sound-mediated physiological or mechanical stress. Such strong impacts suggest that abnormalities and growth delays may also result from lower sound levels or discrete exposures during the D-stage, increasing the potential for routinely-occurring anthropogenic noise sources to affect recruitment of wild scallop larvae in natural stocks.

Trackline and point detection probabilities for acoustic surveys of Cuvier's and Blainville's beaked whales.

Acoustic survey methods can be used to estimate density and abundance using sounds produced by cetaceans and detected using hydrophones if the probability of detection can be estimated. For passive acoustic surveys, probability of detection at zero horizontal distance from a sensor, commonly called g(0), depends on the temporal patterns of vocalizations. Methods to estimate g(0) are developed based on the assumption that a beaked whale will be detected if it is producing regular echolocation clicks directly under or above a hydrophone. Data from acoustic recording tags placed on two species of beaked whales (Cuvier's beaked whale-Ziphius cavirostris and Blainville's beaked whale-Mesoplodon densirostris) are used to directly estimate the percentage of time they produce echolocation clicks. A model of vocal behavior for these species as a function of their diving behavior is applied to other types of dive data (from time-depth recorders and time-depth-transmitting satellite tags) to indirectly determine g(0) in other locations for low ambient noise conditions. Estimates of g(0) for a single instant in time are 0.28 [standard deviation (s.d.) = 0.05] for Cuvier's beaked whale and 0.19 (s.d. = 0.01) for Blainville's beaked whale.

Following a foraging fish-finder: diel habitat use of Blainville's beaked whales revealed by echolocation.

Simultaneous high resolution sampling of predator behavior and habitat characteristics is often difficult to achieve despite its importance in understanding the foraging decisions and habitat use of predators. Here we tap into the biosonar system of Blainville's beaked whales, Mesoplodon densirostris, using sound and orientation recording tags to uncover prey-finding cues available to echolocating predators in the deep-sea. Echolocation sounds indicate where whales search and encounter prey, as well as the altitude of whales above the sea-floor and the density of organisms around them, providing a link between foraging activity and the bio-physical environment. Tagged whales (n = 9) hunted exclusively at depth, investing most of their search time either in the lower part of the deep scattering layer (DSL) or near the sea-floor with little diel change. At least 43% (420/974) of recorded prey-capture attempts were performed within the benthic boundary layer despite a wide range of dive depths, and many dives included both meso- and bentho-pelagic foraging. Blainville's beaked whales only initiate searching when already deep in the descent and encounter prey suitable for capture within 2 min of the start of echolocation, suggesting that these whales are accessing prey in reliable vertical strata. Moreover, these prey resources are sufficiently dense to feed the animals in what is effectively four hours of hunting per day enabling a strategy in which long dives to exploit numerous deep-prey with low nutritional value require protracted recovery periods (average 1.5 h) between dives. This apparent searching efficiency maybe aided by inhabiting steep undersea slopes with access to both the DSL and the sea-floor over small spatial scales. Aggregations of prey in these biotopes are located using biosonar-derived landmarks and represent stable and abundant resources for Blainville's beaked whales in the otherwise food-limited deep-ocean.

Source parameters of echolocation clicks from wild bottlenose dolphins (Tursiops aduncus and Tursiops truncatus).

The Indian Ocean and Atlantic bottlenose dolphins (Tursiops aduncus and Tursiops truncatus) are among the best studied echolocating toothed whales. However, almost all echolocation studies on bottlenose dolphins have been made with captive animals, and the echolocation signals of free-ranging animals have not been quantified. Here, biosonar source parameters from wild T. aduncus and T. truncatus were measured with linear three- and four-hydrophone arrays in four geographic locations. The two species had similar source parameters, with source levels of 177-228 dB re 1 µPa peak to peak, click durations of 8-72 µs, centroid frequencies of 33-109 kHz and rms bandwidths between 23 and 54 kHz. T. aduncus clicks had a higher frequency emphasis than T. truncatus. The transmission directionality index was up to 3 dB higher for T. aduncus (29 dB) as compared to T. truncatus (26 dB). The high directionality of T. aduncus does not appear to be only a physical consequence of a higher frequency emphasis in clicks, but may also be caused by differences in the internal properties of the sound production system.