Kinetic and structural basis of reactivity of pentaerythritol tetranitrate reductase with NADPH, 2-cyclohexenone, nitroesters, and nitroaromatic explosives

The reaction of pentaerythritol tetranitrate reductase with reducing and oxidizing substrates has been studied by stopped-flow spectrophotometry, redox potentiometry, and X-ray crystallography. We show in the reductive half-reaction of pentaerythritol tetranitrate (PETN) reductase that NADPH binds to form an enzyme-NADPH charge transfer intermediate prior to hydride transfer from the nicotinamide coenzyme to FMN. In the oxidative half-reaction, the two-electron-reduced enzyme reacts with several substrates including nitroester explosives (glycerol trinitrate and PETN), nitroaromatic explosives (trinitrotoluene (TNT) and picric acid), and alpha,beta-unsaturated carbonyl compounds (2-cyclohexenone). Oxidation of the flavin by the nitroaromatic substrate TNT is kinetically indistinguishable from formation of its hydride-Meisenheimer complex, consistent with a mechanism involving direct nucleophilic attack by hydride from the flavin N5 atom at the electron-deficient aromatic nucleus of the substrate. The crystal structures of complexes of the oxidized enzyme bound to picric acid and TNT are consistent with direct hydride transfer from the reduced flavin to nitroaromatic substrates. The mode of binding the inhibitor 2,4-dinitrophenol (2,4-DNP) is similar to that observed with picric acid and TNT. In this position, however, the aromatic nucleus is not activated for hydride transfer from the flavin N5 atom, thus accounting for the lack of reactivity with 2,4-DNP. Our work with PETN reductase establishes further a close relationship to the Old Yellow Enzyme family of proteins but at the same time highlights important differences compared with the reactivity of Old Yellow Enzyme. Our studies provide a structural and mechanistic rationale for the ability of PETN reductase to react with the nitroaromatic explosive compounds TNT and picric acid and for the inhibition of enzyme activity with 2,4-DNP.     

Aerobic Degradation of 2,4,6-Trinitrotoluene by Enterobacter cloacae PB2 and by Pentaerythritol Tetranitrate Reductase

Enterobacter cloacae PB2 was originally isolated on the basis of its ability to utilize nitrate esters, such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate, as the sole nitrogen source for growth. The enzyme responsible is an NADPH-dependent reductase designated PETN reductase. E. cloacae PB2 was found to be capable of slow aerobic growth with 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. Dinitrotoluenes were not produced and could not be used as nitrogen sources. Purified PETN reductase was found to reduce TNT to its hydride-Meisenheimer complex, which was further reduced to the dihydride-Meisenheimer complex. Purified PETN reductase and recombinant Escherichia coli expressing PETN reductase were able to liberate nitrogen as nitrite from TNT. The ability to remove nitrogen from TNT suggests that PB2 or recombinant organisms expressing PETN reductase may be useful for bioremediation of TNT-contaminated soil and water.     

Negotiating a noisy, information-rich environment in search of cryptic prey: olfactory predators need patchiness in prey cues

1. Olfactory predator search processes differ fundamentally to those based on vision, particularly when odour cues are deposited rather than airborne or emanating from a point source. When searching for visually cryptic prey that may have moved some distance from a deposited odour cue, cue context and spatial variability are the most likely sources of information about prey location available to an olfactory predator. 2. We tested whether the house mouse (Mus domesticus), a model olfactory predator, would use cue context and spatial variability when searching for buried food items; specifically, we tested the effect of varying cue patchiness, odour strength, and cue-prey association on mouse foraging success. 3. Within mouse- and predator-proof enclosures, we created grids of 100 sand-filled Petri dishes and buried peanut pieces in a set number of these patches to represent visually cryptic 'prey'. By adding peanut oil to selected dishes, we varied the spatial distribution of prey odour relative to the distribution of prey patches in each grid, to reflect different levels of cue patchiness (Experiment 1), odour strength (Experiment 2) and cue-prey association (Experiment 3). We measured the overnight foraging success of individual mice (percentage of searched patches containing prey), as well as their foraging activity (percentage of patches searched), and prey survival (percentage of unsearched prey patches). 4. Mouse foraging success was highest where odour cues were patchy rather than uniform (Experiment 1), and where cues were tightly associated with prey location, rather than randomly or uniformly distributed (Experiment 3). However, when cues at prey patches were ten times stronger than a uniformly distributed weak background odour, mice did not improve their foraging success over that experienced when cues were of uniform strength and distribution (Experiment 2). 5. These results suggest that spatial variability and cue context are important means by which olfactory predators can use deposited odour cues to locate visually cryptic prey. They also indicate that chemical crypsis can disrupt these search processes as effectively as background matching in visually based predator-prey systems.     

Search images not proven: A reappraisal of recent evidence

It is commonly agreed that predators hunting for cryptic prey have been demonstrated to adopt search images (involving perceptual specialization) for particular prey types. However, a simple alternative strategy, that of an appropriate reduction in search rate, may in many cases have the same effect as adopting a search image. The search image and search rate hypotheses are examined, and a series of critical predictions are proposed to distinguish them. Briefly, the search rate hypothesis predicts: (1) that adjusting search rate for one cryptic prey type will enhance the ability to detect other equally cryptic types (the search image hypothesis predicts that this will interfere with the ability to detect other equally cryptic types); (2) that adjusting search rate for cryptic prey will be achieved by learning to spend longer looking at each patch of the environment, and that the more cryptic the prey the longer will be the viewing time required to effect accurate detection (the search image hypothesis makes no such predictions). Four recent studies purporting to demonstrate search images are re-examined in the light of these predictions, and it is found that the published data fail to distinguish the two hypotheses. In addition, some evidence that is better explained by the search rate hypothesis is discussed. A brief examination of the ecological consequences of the search rate hypothesis shows that, unlike the search image hypothesis, it does not predict apostatic selection. It is therefore argued that the distinction between the two mechanisms explaining how predators ‘learn to see’ cryptic prey is important, but that the critical tests have not yet been made. The existence of search images remains not proven.     

The influence of prey-scent stimuli on predatory behavior of the North American copperhead Agkistrodon contortrix (Serpentes: Viperidae)

Viperid snakes strike, envenomate, and release mammalian preyto prevent being harmed by the prey; snakes must then trackprey in the process of strike-induced chemosensory searching.Because rattlesnakes prefer to track and consume envenomatedprey, it would seem that the scent of envenomated tissue iskey to the tracking process. After striking rodents, rattlesnakesalso retain a specific chemical search image of prey items.I examined this behavioral pattern in copperheads (Agkistrodoncontortrix) from three US populations with documented dietarybiases toward mammals (Kansas), lepidopteran larvae (Texas),and amphibians (Louisiana), respectively. Experiments were conductedto assess whether copperheads form a specific search image ofnon-envenomated mouse, hornworm, and frog prey items. Additionalexperiments tested the relative importance of envenomated tissueto prey scent. Results indicate that copperheads do not forma specific search image of prey items. Preference for non-envenomatedprey items is in the order mouse > hornworm > frog forall three populations; therefore, the innate behavioral preferencefor types of prey does not match the dietary biases noted inthe literature. Envenomated mice and hornworms were preferredto all nonenvenomated prey items, but most trials involvingenvenomated frogs did not suggest envenomated prey preference.Overall, these results suggest that when the snakes search forprey, envenomated tissue stimuli are more important to snakesthan scents arising from the prey itself. Searching and consumptionbehaviors seem to be independent, suggesting that strike-inducedchemosensory searching and consumption are more complicatedbehavioral processes than previously recognized.     

Density-dependent predation by skunks using olfactory search images

The formation of search images can create density-dependent predation. Predators have been shown to form search images when searching for many small prey items in one feeding session. This paper reports experiments that test whether striped skunks can form olfactory search images in other situations: when prey are found over several days, when prey are large, and when prey are found in certain habitats. Striped skunks were raised in captivity, and their reaction distance to food was measured outside in a natural grassy area. In experiment 1 skunks were offered many small food items for several days in a row. From one day to the next, skunks initially detected food from further away, they increased detection distance faster and their maximum detection distance increased – i.e., they formed olfactory search images faster and stronger from one day to the next. In experiment 2 skunks formed search images over several days when finding only one large food item per day. In experiment 3 skunks lost olfactory search images when they entered habitats in which they had previously searched for another type of food. These long-term search images magnify the effects of short-term search images, extend the effects of short- term search image to longer time spans, and affect different species from short-term search images. Received: 26 July 1996 / Accepted: 13 December 1996     

Plant processes important for the transformation and degradation of explosives contaminants

Environmental contamination by explosives is a worldwide problem. Of the 20 energetic compounds, 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) are the most powerful and commonly used. Nitroamines are toxic and considered as possible carcinogens. The toxicity and persistence of nitroamines requires that their fate in the environment be understood and that contaminated soil and groundwater be remediated. This study, written as a minireview, provides further insights for plant processes important for the transformation and degradation of explosives. Plants metabolize TNT and the distribution of the transformation products, conjugates, and bound residues appears to be consistent with the green liver model concept. Metabolism of TNT in plants occurs by reduction as well as by oxidation. Reduction probably plays an important role in the tolerance of plants towards TNT, and, therefore a high nitroreductase capacity may serve as a biochemical criterion for the selection of plant species to remediate TNT. Because the activities and the inducibilities of the oxidative enzymes are far lower than of nitroreductase, reducing processes may predominate. However, oxidation may initiate the route to conjugation and sequestration leading ultimately to detoxification of TNT, and, therefore, particularly the oxidative pathway deserves more study. It is possible that plants metabolize RDX also according to the green liver concept. In the case of plant metabolism of HMX, a conclusion regarding compliance with the green liver concept was not reached due to the limited number of available data.     

Evidence for search image in blackbirds (Turdus merula L.): short-term learning

A ‘search image’ is acquired as a result of a change in the ability of a predator to detect cryptic familiar prey. Blackbirds were presented with artificial prey dyed either to match the colour of the background (cryptic prey) or to contrast with the background (conspicuous prey). The results of experiment 1 provide some evidence that 10 wild blackbirds had more difficulty detecting cryptic prey than conspicuous prey. Detailed analysis of experiments 2 (11 wild blackbirds) and 3 (six captive blackbirds) revealed subtle changes in the reactions of the birds to cryptic prey. At first these birds were unable to detect cryptic prey, but subsequently improved their ability to detect such food. This short-term change is interpreted as evidence for search images.     

Phytodetoxification of TNT by transgenic plants expressing a bacterial nitroreductase.

There is major international concern over the wide-scale contamination of soil and associated ground water by persistent explosives residues. 2,4,6-Trinitrotoluene (TNT) is one of the most recalcitrant and toxic of all the military explosives. The lack of affordable and effective cleanup technologies for explosives contamination requires the development of better processes. Significant effort has recently been directed toward the use of plants to extract and detoxify TNT. To explore the possibility of overcoming the high phytotoxic effects of TNT, we expressed bacterial nitroreductase in tobacco plants. Nitroreductase catalyzes the reduction of TNT to hydroxyaminodinitrotoluene (HADNT), which is subsequently reduced to aminodinitrotoluene derivatives (ADNTs). Transgenic plants expressing nitroreductase show a striking increase in ability to tolerate, take up, and detoxify TNT. Our work suggests that expression of nitroreductase (NR) in plants suitable for phytoremediation could facilitate the effective cleanup of sites contaminated with high levels of explosives.     

The effect of learning and search images on predator–prey interactions

In dealing with the spatial and temporal variability of prey species, predators may be able to forage optimally if they have flexible and rapid behavioral plasticity rather than predetermined responses. For predators that learn to focus attention on the cryptic prey type most frequently encountered during recent searching (termed a “search image”), rare prey types may be overlooked because of a focus on more common prey. Search imaging reflects biased searching for one of a number of available prey types, and has been studied widely in birds and mammals. Here we discuss the significant implications of this phenomenon for insect predator–prey systems, particularly with respect to parasitic wasps searching for host species using learned olfactory cues. We (1) review studies about perceptual development through individual ontogeny, (2) define the term “search image” and discuss the cognitive mechanisms involved in search-image formation, (3) discuss the role of search images and frequency-dependent predation as a proximate mechanism in the maintenance of prey diversity, (4) examine data on host–parasitoid olfactory search imaging, and (5) conclude by identifying important research areas for future studies in the field of olfactory search images.     

Prey Detection in Two Tit Species,Parus ater and P. cristatus

We studied the prey-detection response of two related tit species (Parus ater, coal tit, and P. cristatus, crested tit) which occur in similar habitats and which are separated by microhabitat use and mode of locomotion. Specifically, we tested whether the two species differ in their ability to detect cryptic prey, with the hypothesis that coal tits perform better because of their longer stays in microhabitat patches. We expected that interspecific differences and the flexible responses within species to different levels of prey crypticity both depend on search intensity. In the experiments, tits had to forage in four different prey conditions: conspicuous + small, conspicuous + large, cryptic + small, and cryptic + large. Capture success, the time needed to detect prey, and stare duration were measured. Both species were less successful with cryptic than with conspicuous prey and did worst with small and cryptic prey. They also needed more time for detection and stared longer when prey was cryptic. When prey had been overlooked, preceding stare durations were shorter than those ending in successful searches. There were clear differences between the species in performance, with the crested tit doing worse. Coal tits, on average, were more successful in detecting prey, returned less frequently to already emptied patches, detected prey faster and yet they stared more briefly. The within-species results support current notions on predator search, but the differences between species cannot be explained with the same mechanisms. We discuss these findings in the context of the foraging ecology of these species and conclude that crested tits use different cues to detect prey and/or search a larger area per unit time.     

Detecting predator–prey relationships in the sea

Understanding the strength and diversity of predator‐prey interactions among species is essential to understand ecosystem consequences of population‐level variation. Directly quantifying the predatory behaviour of wild fishes at large spatial scales (>100 m) in the open sea is fraught with difficulties. To date the only empirical approach has been to search for correlations in the abundance of predators and their putative prey. As an example we use this approach to search for predators of the keystone crown‐of‐thorns starfish. We show that this approach is unlikely to detect predator–prey linkages because the theoretical relationship is non‐linear, resulting in multiple possible prey responses for single given predator abundance. Instead we suggest some indication of the strength and ecosystem importance of a predator–prey relationship can be gained by using the abundance of both predators and their putative prey to parameterize functional response models.     

Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter.

Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (Km = 23 microM) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35 degrees C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the alpha/beta barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively.     

The evolution of colour polymorphism in British winter‐active Lepidoptera in response to search image use by avian predators

Phenotypic polymorphism in cryptic species is widespread. This may evolve in response to search image use by predators exerting negative frequency‐dependent selection on intraspecific colour morphs, ‘apostatic selection’. Evidence exists to indicate search image formation by predators and apostatic selection operating on wild prey populations, though not to demonstrate search image use directly resulting in apostatic selection. The present study attempted to address this deficiency, using British Lepidoptera active in winter as a model system. It has been proposed that the typically polymorphic wing colouration of these species represents an anti‐search image adaptation against birds. To test (a) for search image‐driven apostatic selection, dimorphic populations of artificial moth‐like models were established in woodland at varying relative morph frequencies and exposed to predation by natural populations of birds. In addition, to test (b) whether abundance and degree of polymorphism are correlated across British winter‐active moths, as predicted where search image use drives apostatic selection, a series of phylogenetic comparative analyses were conducted. There was a positive relationship between artificial morph frequency and probability of predation, consistent with birds utilizing search images and exerting apostatic selection. Abundance and degree of polymorphism were found to be positively correlated across British Lepidoptera active in winter, though not across all taxonomic groups analysed. This evidence is consistent with polymorphism in this group having evolved in response to search image‐driven apostatic selection and supports the viability of this mechanism as a means by which phenotypic and genetic variation may be maintained in natural populations.     

Sensory basis for detection of benthic prey in two Lake Malawi cichlids

The adaptive radiations of African cichlids resulted in a diversity of feeding morphologies and strategies, but the role of sensory biology in prey detection and feeding ecology remains largely unexplored. Two endemic Lake Malawi cichlid genera, Tramitichromis and Aulonocara, feed on benthic invertebrates, but differ in lateral line morphology (narrow and widened lateral line canals, respectively) and foraging strategy. The hypothesis that they use their lateral line systems differently was tested by looking at the relative contribution of the lateral line system and vision in prey detection by Tramitichromis sp. and comparing results to those from a complementary study using Aulonocara stuartgranti (Schwalbe et al., 2012). First, behavioral trials were used to assess the ability of Tramitichromis sp. to detect live (mobile) and dead (immobile) benthic prey under light and dark conditions. Second, trials were run before, immediately after, and several weeks after chemical ablation of the lateral line system to determine its role in feeding behavior. Results show that Tramitichromis sp. is a visual predator that neither locates prey in the dark nor depends on lateral line input for prey detection and is thus distinct from A. stuartgranti, which uses its lateral line or a combination of vision and lateral line to detect prey depending on light condition. Investigating how functionally distinctive differences in sensory morphology are correlated with feeding behavior in the laboratory and determining the role of sensory systems in feeding ecology will provide insights into how sensory capabilities may contribute to trophic niche segregation.     

The role of learning by a predator,Rivulus hartii,in the rare‐morph survival advantage in guppies

Negative frequency‐dependent selection (NFDS), where rare types are favoured by selection, can maintain diversity. However, the ecological processes that mediate NFDS are often not known. Male guppies (Poecilia reticulata) exhibit extreme diversity of colour patterning and, in a previous field experiment, rare morphs had a survival advantage. Here, we test the hypothesis that predators impose NFDS because they are efficient at capturing familiar prey morphs, but are less efficient at capturing unfamiliar morphs. Over a series of trials, we presented Rivulus hartii, a natural predator of guppies, with male guppies with the same colour patterning (A trials); then, for a second series of trials, we presented the rivulus with guppies with a new colour pattern (B trials). The success of rivulus at capturing guppies on the first attack increased over successive A trials. First attack success decreased significantly for the early B trials, and then increased during successive B trials, eventually reaching the same level as in the best A trials. This experiment demonstrates that learning, perhaps through long‐term search image formation, plays a role in predation success on familiar vs. unfamiliar prey morphs. These results support the hypothesis that predator learning contributes to the maintenance of the extreme male guppy polymorphism seen in nature.     

Demographic and functional responses of wild dogs to poison baiting

Lethal control of wild dogs – that is Dingo (Canis lupus dingo) and Dingo/Dog (Canis lupus familiaris) hybrids – to reduce livestock predation in Australian rangelands is claimed to cause continental‐scale impacts on biodiversity. Although top predator populations may recover numerically after baiting, they are predicted to be functionally different and incapable of fulfilling critical ecological roles. This study reports the impact of baiting programmes on wild dog abundance, age structures and the prey of wild dogs during large‐scale manipulative experiments. Wild dog relative abundance almost always decreased after baiting, but reductions were variable and short‐lived unless the prior baiting programme was particularly effective or there were follow‐up baiting programmes within a few months. However, age structures of wild dogs in baited and nil‐treatment areas were demonstrably different, and prey populations did diverge relative to nil‐treatment areas. Re‐analysed observations of wild dogs preying on kangaroos from a separate study show that successful chases that result in attacks of kangaroos by wild dogs occurred when mean wild dog ages were higher and mean group size was larger. It is likely that the impact of lethal control on wild dog numbers, group sizes and age structures compromise their ability to handle large difficult‐to‐catch prey. Under certain circumstances, these changes sometimes lead to increased calf loss (Bos indicus/B. taurus genotypes) and kangaroo numbers. Rangeland beef producers could consider controlling wild dogs in high‐risk periods when predation is more likely and avoid baiting at other times.     

Sex‐ and age‐related differences in c‐fos expression in dog olfactory bulbs

Sex‐ and age‐related differences in cognitive abilities are frequently reported. However, the sex‐ and age‐related differences in dog olfaction due to biological system are still poorly understood. We examined c‐fos expression in dog olfactory bulbs by immunohistochemistry approaches. The c‐fos is mainly expressed in the olfactory glomerular layer (GL), mitral cell layer (ML) and granule cell layer (GRL). We found that a higher density of c‐fos‐positive cells could be detected in the ML of olfactory bulbs of adult female dogs compared with that in males and the c‐fos‐positive cells in females' olfactory bulbs are more distinct. Sex‐related differences in c‐fos expression also appeared in the GL of olfactory bulbs in juvenile dogs. We also discovered that the density of c‐fos‐positive cells in the GRL of adult dogs was much higher than that in the GRL of juvenile dogs. Our results indicate that cells in the olfactory bulbs of female dogs are more active than those in males and female dogs may have much stronger ability for long‐time memory of odours than male dogs. Furthermore, our results also suggest that adult dogs may have much stronger ability for long‐time memory of odours and can deal with more complicated odour information than juvenile dogs.     

Effect of plant extract on the degradation of nitroaromatic compounds by soil microorganisms

Remediation of soils contaminated by nitroaromatic compounds and nitramines, i.e. explosives, is known as very important, complicated, and rapidly developing area of biotechnology. A search for optimal growth conditions for soil bacteria is of a great importance in order to isolate various xenobiotic degraders. Bacteria consortium A43 was isolated from soils contaminated with explosives. In the presence of carbohydrate and plant extract, an addition of TNT to the solidified minimal medium stimulated the growth of the tested bacteria, as compared to other bacteria consortium isolated from the same soils. Reducing sugars as carbohydrates, and cabbage leaf extract as a plant extract were used in these experiments. Cultivation of the A43 in liquid medium of the same content showed that addition of cabbage leaf extract alone to medium is much more efficient for TNT degradation by growing biomass as compared to addition of carbohydrate alone.     

Are dogs able to recognize their handler's voice? A preliminary study

Abstract

A number of studies have dealt with the ability of dogs to rely on olfactory cues to recognize their owner or handler. However, in the absence of these cues (e.g., when working at a distance from their owner) dogs have to use other senses to interact with their owner. Considering the high sensitivity of their auditory system, we can hypothesize that dogs are able to use vocal cues for this purpose. Certainly, it is commonly assumed that dogs can discriminate between people on the basis of their voice, and yet there is a clear lack of empirical data on this subject. The present study assesses the importance of the handler's olfactory and visual cues versus the importance of his/her vocal cues to the working behavior of dogs. I compare the ability of working dogs to obey different vocal commands issued either by their handler (HV) or by a stranger (SV) via loudspeakers in different olfactory contexts: presence of their handler (H) or of a stranger (S). The dogs' performance (percentage of correct responses) decreased when the acoustic cues were modified (H+HV tended to differ from the H+SV—p = 0.09—and differed from the S+SV conditions—p = 0.05) but not when only the olfactory cues were changed (no difference between H+HV and S+HV conditions—p > 0.1). Consequently, we can assume that dogs rely, at least in part, on acoustic parameters of voices to recognize and cooperate with their handler.