Testing the differential predation hypothesis for the invasion of rusty crayfish in a stream community: laboratory and field experiments

1. We tested the hypothesis that the non‐native rusty crayfish (Orconectes rusticus) is less vulnerable to predators than two native species (O. propinquus and O. obscurus) it is replacing in streams of the upper Susquehanna River catchment (New York, U.S.A.). 2. We used laboratory experiments to compare species‐specific predation rates by smallmouth bass (Micropterus dolomieu) on crayfish of equal size and field tethering experiments to compare relative predation rates between native O. propinquus and non‐native O. rusticus by the suite of crayfish predators in our system. We predicted that crayfish size would affect predation rate but that predation rates would be equal among species when size was controlled. 3. We also tested for two potential artefacts of tethering. We tethered crayfish in cages to test whether the ability to escape from tethers is size specific, and we tested whether tethering alters differential predation among crayfish species by the smallmouth bass. 4. In the laboratory, smallmouth bass predation on rusty crayfish was lower than on either of the native species. In the field, predation risk for tethered crayfish was inversely related to size but did not differ among species when size was taken into account. Because rusty crayfish in the field experiment were slightly larger than the native species, as in nature, mortality was overall lower for the rusty crayfish. 5. In cages, smaller crayfish were more probably to escape from tethers than larger ones, an artefact that may partially confound results from our tethering experiments. Unexpectedly, tethering nearly eliminated predation by smallmouth bass. This artefact prevented us from testing for an interaction of tethering with differential predation and means that the results of field tethering experiments do not include any contribution from smallmouth bass predation. 6. Our experiments highlight the importance of explicitly considering potential artefacts that could confound results. 7. Our results indicate that differential predation contributes to the rusty crayfish's invasion of a stream community. In our study system, predation rates on rusty crayfish are lower than for native species mostly because of selection by predators for smaller crayfish; species‐specific characteristics such as behaviour that further reduce predation may also contribute, especially where smallmouth bass predation is important.     

Ontogeny of swimming speed, schooling behaviour and jellyfish avoidance by Japanese anchovy Engraulis japonicus

The ontogeny of swimming speed, schooling behaviour and jellyfish avoidance was studied in hatchery-reared Japanese anchovy Engraulis japonicus to compare its life-history strategy with two other common pelagic fishes, jack mackerel Trachurus japonicus and chub mackerel Scomber japonicus. Cruise swimming speed of E. japonicus increased allometrically from 1·4 to 3·9 standard length (L(S) ) per s (L(S) s(-1) ) from early larval to metamorphosing stage. Burst swimming speed also increased from 6·1 to 28 L(S) s(-1) in these stages. Cruise speed was inferior to that of S. japonicus, as was burst speed to that of T. japonicus. Engraulis japonicus larvae were highly vulnerable to predation by moon jellyfish Aurelia aurita and were readily eaten until they reached 23 mm L(S) , but not at 26 mm L(S) . Schooling behaviour (indicated by parallel swimming) started at c. 17 mm L(S) . Average distance to the nearest neighbour was shorter than values reported in other pelagic fishes. The relatively low predator avoidance capability of E. japonicus may be compensated for by their transparent and thus less conspicuous body, in addition to their early maturation and high fecundity.     

Behavioural mechanisms of sea stars (Asterias vulgaris Verrill and Leptasterias polaris Müller) and crabs (Cancer irroratus Say and Hyas araneus Linnaeus) preying on juvenile sea scallops (Placopecten magellanicus (Gmelin)), and procedural effects of scallop tethering

We compared predation rates and behaviours of sea stars (Asterias vulgaris and Leptasterias polaris) and crabs (Cancer irroratus and Hyas araneus) preying on juvenile sea scallops (Placopecten magellanicus, 25-35 mm shell height) in laboratory. These predatory species co-occur with sea scallops on the sea bed of the Gulf of St. Lawrence, Canada, and limit scallop survival in seeding operations. We also examined, under controlled conditions, the effect of tethering scallops on predator-prey interactions. Predation rates, time budgets and encounter behaviours observed for A. vulgaris and C. irroratus preying on free (untethered) scallops were comparable to previous studies. C. irroratus were more effective predators as they consumed 3.1 scallops predator^− 1 day^− 1, although they spent only 0.9% of their time searching for prey. A. vulgaris consumed 0.9 scallops predator^− 1 day^− 1 and spent 7.6% of their time searching. Sea stars L. polaris had a lower predation rate (0.02 scallop predator^− 1 day^− 1) than A. vulgaris. The frequent avoidance behaviour of L. polaris and its low ability to capture scallops support the notion that scallops are not a main component of this sea star's diet. Crabs H. araneus had similar predation rates (1.3 scallops predator^− 1 day^− 1) and behaviours to C. irroratus, although the probability of consumption upon capture was affected by relatively high numbers of rejections and post-capture escapes of scallops. As expected, the tethering procedure increased predation rate of L. polaris (about 19 times higher), but surprisingly did not significantly affect that of A. vulgaris. Examination of behaviours indicated that A. vulgaris offered tethered scallops tended to have a higher probability of capture, but spent less time searching for prey (possibly because satiation was reached) than A. vulgaris offered free scallops. Predation rates and behaviours of both crab species were not affected by tethering, since encounter rate was the primary determinant of crab-scallop interactions. Identification and quantification of behaviours underlying the predation process allowed us to mathematically model predator-related mortality for the four predator species.     

Release method and anatomical hook location: effects on short-term mortality of angler-caught Acanthopagrus australis and Argyrosomus japonicus

One field and 3 aquaria experiments were done to quantify the short-term mortality of yellowfin bream Acanthopagrus australis and mulloway Argyrosomus japonicus after being angled and subjected to 3 general handling treatments. Anglers were supplied with identical J-type hooks and asked to handle hooked fish by either (1) physically removing the hook or (2) cutting the line (5 cm from the mouth of the fish) and leaving the hook in. Some hooked A. japonicus were subjected to a third handling treatment where the line was cut underwater without exposing the fish to air. Technical and biological data were collected before all fish were released into sea cages and monitored for 5 d. Control fish were seined and similarly caged and monitored. Concentrations of plasma glucose and cortisol were collected from a sample of fish on the first and last day of the experiments. Significant predictors of mortality for both species involved the presence of blood at the mouth and an interaction between anatomical hook location and hook removal. A. australis and A. japonicus that had their ingested hooks removed experienced the greatest mortalities (87.5 and 72.7%, respectively). Typically, these fish suffered damage to their oesophagus, stomach wall and vital organs. Mortality rates of A. australis and A. japonicus were significantly decreased to 1.7 and 16%, respectively, when they were released with their lines cut, with some of these fish free of hooks after 5 d. In contrast, few mortalities occurred in either species when the hooks were removed or the lines cut on mouth-hooked fish or in A. japonicus when it was released with no air exposure. For A. australis, the field- and aquaria-based experiments provided comparable results in terms of identifying treatment-specific effects, but there were potential biases in rates of hook ingestion. Irrespective of the treatment of fish, all experiments caused physiological changes measured as elevations in either plasma cortisol or glucose. We concluded that anglers should cut the line from hook-ingested A. australis and A. japonicus, but remove the hook from mouth-hooked individuals to prevent subsequent ingestion. Further research is required to examine the longer-term consequences of these handling practices on fish health.     

Roles of spatial partitioning,competition, and predation in the North American invasion of an exotic mosquito

Invasion success and species coexistence are often mediated by species interactions across patchily distributed habitats and resources. The invasive mosquito Aedes japonicus japonicus has established in the North American range of the competitively superior resident congener, Aedes albopictus, and the predatory native mosquito Toxorhynchites rutilus. We tested predictions for two hypotheses of invasion success and species coexistence: keystone predation and spatial partitioning. We tested competition between A. japonicus japonicus and A. albopictus with or without T. rutilus in laboratory microcosms, and measured abundances of A. japonicus japonicus, A. albopictus, other resident competing mosquito species, and the presence of T. rutilus among tree holes and tires in metropolitan Washington, DC. In laboratory microcosms, A. albopictus was competitively dominant over A. japonicus japonicus, which is consistent with the few prior studies of competition between these two Aedes species. T. rutilus predation severely lowered performances of both Aedes species but more severely lowered A. japonicus japonicus performance than A. albopictus performance when all three species co-occurred, thus yielding no evidence for keystone predation. Consistent with the spatial partitioning hypothesis, A. japonicus japonicus was negatively correlated and independently aggregated with A. albopictus and all combined resident mosquito competitors and was not associated with T. rutilus among field containers. These results suggest that predation from T. rutilus and competition from A. albopictus are barriers to the spread of A. japonicus japonicus, but that A. japonicus japonicus may escape these interspecific effects by utilizing spatially partitioned container habitats.     

Influence of rearing and experimental temperatures on predator avoidance behaviour in a freshwater pulmonate snail

1. Predicted increases in the temperature of freshwaters is likely to affect how prey species respond to predators. We investigated how the predator avoidance behaviour of the freshwater pulmonate snail Lymnaea stagnalis is influenced by the temperature at which it was reared and that at which behavioural trials were carried out. 2. Crawl‐out behaviour of juvenile snails from two populations (high predation risk versus low predation risk) reared at either 15 or 20 °C was assessed in response to predation cues (predatory fish kairomones and conspecific alarm cues) in behavioural trials at both 15 and 20 °C. 3. Trial temperature had a significant effect on the time that snails spent in avoidance, regardless of their population of origin. Crawl‐out behaviour was greater during behavioural trials at 15 °C, but there was no effect of trial temperature on the speed with which animals showed avoidance behaviour. 4. There was no interactive effect of rearing temperature (RT) and trial temperature, but the effect of RT on avoidance behaviour did differ between populations. For an RT of 15 °C, snails from the South Drain (high risk) population showed a more rapid and longer avoidance response than those from the Chilton Moor (low risk) population. In contrast, for snails reared at 20 °C, there was no difference between populations for the duration of the avoidance response and snails from Chilton Moor crawled out faster than those from South Drain. 5. Hence, whilst (predictable) differences relative to natural predation threat in crawl‐out behaviour were apparent at 15 °C, raising the developmental temperature to 20 °C eliminated or, in the case of latency, reversed these differences. This suggests that L. stagnalis populations that cohabit with predatory fish and experience high developmental temperatures may have a reduced ability to respond to fish predation risk.     

Characteristics of palinurids (Decapoda; Crustacea) in larval culture

Complete development of phyllosomas wasaccomplished in Palinurus elephas, Panulirus japonicus, Jasus edwardsii and J. verreauxi. Larval development differsbetween species in duration and moult frequency aswell as morphology. The highest percent survival was obtained during the phyllosoma stage forJ. verreauxi. Phyllosomas of P. elephas were the mostdifficult to cultureregardless of the fact that they hatch at arelatively advanced stage. Culture experiments werecarried out to improve the percent survival of P. elephas. First instars of P. elephasexhibited vigorous predation upon the larvae ofJapanese sandfish Arctoscopus japonicus.Duration of the first instar was shorter at 12.8days when reared at 18 °C and percent survival wasapproximately 30%. Better results were obtainedfor first instars cultured in water inoculated withChaetoceros sp. and fed enriched Artemianauplii. Phyllosomas of J. edwardsii and P. japonicus were cultured without particulardifficulty. Most distinguishing features of P. elephasis the heavily setose six or seven longspines on the distal edges of the scaphognathite ofthe second maxillae while only four long spines arefound in other species. These facts suggest thatphyllosomas of P. elephas are initiallyplankton feeders and from first instar becomerapacious predators.     

The effect of predation risk on the predatory behaviour of a sit-and-wait predator,Ranatra dispar (Heteroptera: Nepidae), the water stick insect

Two primary defence behaviours, fore-leg extension to enhance crypsis and swimming to bottom and remaining motionless, of a predatory water bug,Ranatra dispar, are described along with their subsequent effect on foraging behaviour. It was hypothesised that hungry predators would respond less and for a shorter duration compared with recently fed individuals when exposed to a model threat stimulus, thereby tending to take a higher risk of predation during feeding than satiated animals. A greater proportion of animals responded overall with the leg extension response compared with the swimming response, although the mean duration of the former was significantly shorter than the latter response. A significantly higher proportion of nonfasted predators responded, and for a longer duration than fasted individuals. The type of model used significantly effected the proportion of animals that responded with leg extensions but not on its duration. In contrast, both number of animals and the duration of the swim/motionless behaviour were significantly effected by stimulus type. The subsequent effect of these 2 behavioural responses on feeding behaviour was examined and showed that although about the same number of predators removed prey from their mouthparts during both responses, significantly more prey were dropped, and therefore lost, during swimming. The results clearly indicate the significant effects that defensive behaviours have on time budgets in foraging behaviour.     

Ontogeny of antipredator performance in hatchery-reared Japanese anchovy Engraulis japonicus larvae exposed to visual or tactile predators in relation to turbidity

Laboratory experiments revealed distinct effects of turbidity on the survival of Japanese anchovy Engraulis japonicus larvae when exposed to either visual (jack mackerel Trachurus japonicus) or tactile (moon jellyfish Aurelia aurita) predators. The experiments were conducted in 30 l tanks with three levels of turbidity obtained by dissolving 0, 50 or 300 mg l(-1) of kaolin. Predators were introduced to experimental tanks followed by larvae of E. japonicus ranging from 5 to 25 mm standard lengths (L(s) ). When exposed to T. japonicus, the mean survival rate of larvae was significantly higher in 300 mg l(-1) treatments compared to the other turbidity levels. When exposed to A. aurita, however, there was no difference in the survival rates among different turbidity treatments. The survival rates when exposed to either predator improved with larval growth. The logistic survivorship models for E. japonicus larvae when exposed to A. aurita had an inflection point at c. 12 mm L(s) , suggesting that their size refuge from A. aurita is close to this value. Comparison to a previous study suggests a high vulnerability of shirasu (long and transparent) fish larvae to jellyfish predation under turbidity. This study indicates that anthropogenic increases of turbidity in coastal waters may increase the relative effect of jellyfish predation on fish larvae.     

Adaptive significance of death feigning posture as a specialized inducible defence against gape-limited predators

Death feigning is fairly common in a number of taxa, but the adaptive significance of this behaviour is still unclear and has seldom been tested. To date, all proposed hypotheses have assumed that prey manage to escape predation by sending a death-mimicking signal, although death-feigning postures are markedly different from those of dead animals. Moreover, the efficacy of this technique may largely depend on the foraging mode of the predator; death feigning seldom works with sit-and-wait predators that make the decision to attack and consume prey within a very brief time. We examined whether death feigning in the pygmy grasshopper Criotettix japonicus Haan was an inducible defence behaviour against the frog Rana nigromaculata, a sit-and-wait, gape-limited predator. The characteristic posture assumed by the grasshopper during death feigning enlarges its functional body size by stretching each of three body parts (pronotum, hind legs and lateral spines) in three different directions, thereby making it difficult for the predator to swallow the prey. Our result is the first consistent explanation for why death-mimicking animals do not always mimic the posture of dead animals.     

Reducing stem bending increases the height growth of tall pines

The hypothesis was tested that upper limits to height growth in trees are the result of the increasing bending moment of trees as they grow in height. The increasing bending moment of tall trees demands increased radial growth at the expense of height growth to maintain mechanical stability. In this study, the bending moment of large lodgepole pine (Pinus contorta Dougl. Ex Loud. var. latifolia Engelm.) was reduced by tethering trees at 10 m height to counter the wind load. Average bending moment of tethered trees was reduced to 38% of control trees. Six years of tethering resulted in a 40% increase in height growth relative to the period before tethering. By contrast, control trees showed decreased height growth in the period after tethering treatment. Average radial growth along the bole, relative to height growth, was reduced in tethered trees. This strongly suggests that mechanical constraints play a crucial role in limiting the height growth of tall trees. Analysis of bending moment and basal area increment at both 10 m and 1.3 m showed that the amount of wood added to the stem was closely related to the bending moment produced at these heights, in both control and tethered trees. The tethering treatment also resulted in an increase in the proportion of latewood at the tethering height, relative to 1.3 m height. For untethered control trees, the ratio of bending stresses at 10 m versus 1.3 m height was close to 1 in both 1998 and 2003, suggesting a uniform stress distribution along the outer surface of the bole.     

Inspection behaviour and inter-individual cooperation in juvenile qingbo: the effects of prior predator exposure and food deprivation

The predation pressure and food availability to which individuals are exposed during their life histories shape inspection behaviour in animals. In this study, we aimed to test whether such behaviours varied with prior experience (predation, starvation or both treatments) or measurement condition (with or without the presence of a predator; here, the snakehead fish, Channa argus) in the fish species Spinibarbus sinensis, known as qingbo. Unexpectedly, prior predator experience showed no significant effect on inspection behaviour as demonstrated by either the frequency or the duration of each activity outside shelter or on cooperation as demonstrated by the inter-individual distance or synchronization of speed. This may have been due to the different adjustments in behaviour among individuals (more shelter use vs. more inspection), the predator treatment used in the present study (exposure to caged predator rather than direct predation) and/or a species-specific strategy in the qingbo. The starved fish displayed shorter inspection latency, increased inspection behaviour and greater cooperation when measured without the predator; however, when measured in the presence of the predator, the starved fish showed increased inspection frequency but shorter inspection duration, possibly due to the compromise between energy needs and predation risk. Similar to those of the predation group, the fish from the double-treated group showed no difference in inspection behaviour compared to the control group under the predator-absent condition, while the high-frequency, short-duration inspection behaviours remained the same as in the starved group. These findings suggested that the adjustment of inspection behaviour and related cooperation are rather complicated according to either predator experience or food deprivation, partially due to the inter-individual differences in behavioural adjustment and/or different environmental conditions.     

Habitat-specific effects of particle size, current velocity, water depth, and predation risk on size-dependent crayfish distribution

This study assessed effects of abiotic (current velocity, water depth, particle size) and biotic (predation risk for crayfish, size distribution and densities of predatory fish) variables on habitat- and size-specific distribution patterns of lotic crayfish (Orconectes obscurus) using field surveys and tethering experiments. Additionally, particle size manipulations were used with predation assays to assess habitat-specific interactions since the average particle size increased from deep pools to shallow pools to riffles. Large crayfish had the highest densities in deep pools and were associated with increased water depth, whereas small and medium crayfish had the highest densities in shallow pools and were strongly associated with increased particle size and decreased water depth. Regardless of size, crayfish in deep pools had significantly lower survival than in shallow pools and riffles. However, only small crayfish showed consistent differences in predation risk by habitat type and were significantly more vulnerable to predation than larger crayfish. Additionally, large rocky refugia resulted in significantly higher survival of small crayfish in the combined particle manipulation/tethering experiment. Overall, predation appears to be a key mechanism structuring habitat-specific distribution patterns for only small O. obscurus. Large substrates may be particularly important in habitats where both small crayfish density and predation risk are high.     

根田鼠母体捕食应激对其雄性子代性行为去雄性化的效应

母体应激效应是母体在妊娠期经历的环境变化对子代发育、繁殖及存活等特征的影响(MousseauandFox ,1 998;Bernado ,1 996 )。该效应不仅能使子代生活史特征产生迟滞性效应(Beckermanetal.,2 0 0 2 ) ,而且在密度制约过程中,导致对种群的迟滞性密度制约(Delayeddensitydependenc     

Risk of predation as a promoting factor of species divergence in threespine sticklebacks (Gasterosteus aculeatus L.)

Icelandic freshwater systems are geologically young and contain only six species of freshwater fish. As these species colonized Icelandic fresh waters they were presented with a diversity of unique, uncontested habitats and food resources, promoting the evolution of new behaviour strategies crucial to the formation of new morphs and speciation. To determine the likelihood that predation threat could affect the antipredator behaviour and possibly the sympatric divergence of prey populations, we analysed antipredator behaviour of seven groups of Icelandic threespine sticklebacks ( Gasterosteus aculeatus ): two marine groups, one group from a lake without piscine predators, and two polymorphic lake populations, each with two groups occupying unique habitats. Shoaling cohesion, school formation and duration, and vigilance in predator inspection/avoidance behaviour varied greatly among groups. The differences appeared to be related to the risk of predation as well as to opportunities and constraints set by the different habitats. Antipredator behaviour was especially pronounced and differed extensively in two polymorphic forms from the lake Thingvallavatn, where predation risk is very high. By keeping the two morphs separate in their respective habitats, high predation risk may be a contributing factor in promoting the habitat-specific divergence of G. aculeatus seen in the lake. This suggests that in situations where refuge habitats are spatially separated, the risk of predation may contribute to the evolution of separate sympatric forms of small fish such as G. aculeatus .  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 189–203.     

Does predation risk influence habitat use by northern redbelly dace Phoxinus eos at different spatial scales?

This study investigated the relationship between spatial variations in predation risk and abundance of northern redbelly dace Phoxinus eos at both macroscale (littoral v. pelagic zones) and microscale (structured v. open water habitats in the littoral zone) of Canadian Shield lakes. Minnow traps were placed in both structured and open water habitats in the littoral zone of 13 Canadian Shield lakes, and estimates of the relative predation risk of P. eos in both the pelagic and the littoral zones were obtained from tethering experiments. Results showed that (1) the mean abundance of P. eos in the littoral zone was positively correlated with the relative predation risk in the pelagic zone, (2) P. eos preferentially used structured over open water habitats in the littoral zone and (3) this preference was not related to the relative predation risk in the littoral zone but decreased as the relative predation risk increased in the pelagic zone. At the lake level, these results support the hypothesis that P. eos enter the littoral zone to avoid pelagic piscivores. At the littoral zone level, the results do not necessarily contradict the widely accepted view that P. eos preferentially use structured over open habitats to reduce their predation risk, but suggest that flexibility in antipredator tactics ( e.g. shelter use v. shoaling) could explain the spatial distribution of P. eos between structured and open water habitats.     

Experimental evidence for innate predator recognition in the Seychelles warbler.

Nest predation is a major determinant of fitness in birds and costly nest defence behaviours have evolved in order to reduce nest predation. Some avian studies have suggested that predator recognition is innate whereas others have stressed the importance of learning. However, none of these studies controlled for the genetic origin of the populations investigated and the effect of unfamiliarity with the predator. Here we determined whether experience with a nest predator is a prerequisite for nest defence by comparing predator recognition responses between two isolated but genetically similar Seychelles warbler (Acrocephalus sechellensis) populations, only one of which had experience of the egg predating Seychelles fody (Foudia sechellarum). Individuals in the predator-free population significantly reduced nest guarding compared to individuals in the population with the predator, which indicates that this behaviour was adjusted to the presence of nest predators. However, recognition responses (measured as both alarm call and attack rates) towards a mounted model of the fody were equally strong in both populations and significantly higher than the responses towards either a mounted familiar non-predator and a mounted, novel, non-predator bird species. Responses did not differ with a warbler's age and experience with the egg predator, indicating that predator recognition is innate.     

Anti-predation behaviour during bird migration; the benefit of studying multiple behavioural dimensions

Predation and predation risk have recently been shown to have profound effects on bird migration, but we still know relatively little about how birds respond to predation risk en route and how this is translated into fundamental aspects of optimal migration. Here, we make the case that to understand the fitness consequences of foraging and anti-predation behaviour en route we cannot rely on single behaviour relationships but must take many aspects of behaviour into account, because of predation risk compensation. We show this in a case study of fat and vigilant birds feeding close to cover, which emphasises the importance and potential of predation risk compensation. Another reason for taking many aspects of behaviour into account is that different behaviours need not contribute equally to individual fitness. Birds faced with an increased predation risk during migration can compensate for increased predation risk in different ways. This implies that the adaptive value of a behavioural trait can still be ambiguous even if a survival cost can be correlated with particular behaviour where all other things are equal (e.g. in an experiment). That is because in natural systems there may frequently be many other ways for animals to compensate, because all other things are never equal, so that the particular behaviour can actually be of little consequence to individual fitness. In conclusion, when studying foraging decisions and anti-predation behaviour during stopover potential compensatory mechanisms should be incorporated. This knowledge is also critical for improving future models of optimal migration.     

Enhanced Tethered-Particle Motion Analysis Reveals Viscous Effects

Tethered-particle motion experiments do not require expensive or technically complex hardware, and increasing numbers of researchers are adopting this methodology to investigate the topological effects of agents that act on the tethering polymer or the characteristics of the polymer itself. These investigations depend on accurate measurement and interpretation of changes in the effective length of the tethering polymer (often DNA). However, the bead size, tether length, and buffer affect the confined diffusion of the bead in this experimental system. To evaluate the effects of these factors, improved measurements to calibrate the two-dimensional range of motion (excursion) versus DNA length were carried out. Microspheres of 160 or 240 nm in radius were tethered by DNA molecules ranging from 225 to 3477 basepairs in length in aqueous buffers containing 100 mM potassium glutamate and 8 mM MgCl₂ or 10 mM Tris-HCl and 200 mM KCl, with or without 0.5% Tween added to the buffer, and the motion was recorded. Different buffers altered the excursion of beads on identical DNA tethers. Buffer with only 10 mM NaCl and >5 mM magnesium greatly reduced excursion. Glycerol added to increase viscosity slowed confined diffusion of the tethered beads but did not change excursion. The confined-diffusion coefficients for all tethered beads were smaller than those expected for freely diffusing beads and decreased for shorter tethers. Tethered-particle motion is a sensitive framework for diffusion experiments in which small beads on long leashes most closely resemble freely diffusing, untethered beads.     

Entropic benefit of a cross-link in protein association

We introduce a method to estimate the loss of configurational entropy upon insertion of a cross-link to a dimeric system. First, a clear distinction is established between the loss of entropy upon tethering and binding, two quantities that are often considered to be equivalent. By comparing the probability distribution of the center-to-center distances for untethered and cross-linked versions, we are able to calculate the loss of translational entropy upon cross-linking. The distribution function for the untethered helices is calculated from the probability that a given helix is closer to its partner than to all other helices, the "Nearest Neighbor" method. This method requires no assumptions about the nature of the solvent, and hence resolves difficulties normally associated with calculations for systems in liquids. Analysis of the restriction of angular freedom upon tethering indicates that the loss of rotational entropy is negligible. The method is applied in the context of the folding of a ten turn helical coiled coil with the tether modeled as a Gaussian chain or a flexible amino acid chain. After correcting for loop closure entropy in the docked state, we estimate the introduction of a six-residue tether in the coiled coil results in an effective concentration of the chain to be about 4 or 100 mM, depending upon whether the helices are denatured or pre-folded prior to their association. Thus, tethering results in significant stabilization for systems with millimolar or stronger dissociation constants.