Hippotherium primigenium, with moderately hypsodont cheek teeth, rapidly dispersed through Eurasia in the early late Miocene. This dispersal of hipparions into the Old World represents a major faunal event during the Neogene. The reasons for this fast dispersal of H. primigenium within Europe are still unclear. Based on its hypsodonty, a high specialization in grazing is assumed although the feeding ecology of the earliest European hipparionines within a pure C3 plant ecosystem remains to be investigated.The equid H. primigenium populations from four early Late Miocene localities in Germany , Switzerland (Charmoille), and France (Soblay). Enamel δ13C values indicate a pure C3 plant diet with small (<1.4‰) seasonal variations for all four H. primigenium populations. Dental wear and carbon isotope compositions are compatible with dietary differences. Except for the Höwenegg hipparionines, dental microwear data indicate a browse-dominated diet. By contrast, the tooth mesowear patterns of all populations range from low to high abrasion suggesting a wide spectrum of food resources.A multi-proxy approach, combining carbon and oxygen isotopes from enamel as well as dental meso- and microwear analyses of cheek teeth, was used to characterize the diet of the earliest European 3 ecosystems. Different H. primigenium populations in Europe had a large spectrum of feeding habits with a high browsing component. The combination of specialized phenotypes such as hypsodont cheek teeth with a wide spectrum of diet illustrates a new example of the Liem’s paradox. This dietary flexibility associated with the capability to exploit abrasive food such as grasses probably contributed to the rapid dispersal of hipparionines from North America into Eurasia and the fast replacement of the brachydont equid Anchitherium by the hypsodont H. primigenium in Europe.Combined dental wear and stable isotope analysis enables refined palaeodietary reconstructions in C The first occurrence of high-crowned tridactyl hipparionine horses in western Eurasia (‘Hipparion’ Datum) has recently been pushed back to the early Late Miocene  with discovery of Hippotherium primigenium in Atzelsdorf, Austria 3 grasslands were actually widespread from the Middle Miocene, prior the first occurrence of hipparionines in the Old World 4 monocotyledons did not radiate across Western Eurasia during the Neogene Neogene Old World equids are all derived from a North American common ancestor that dispersed into Eurasia across Beringia during times of glacio-eustatic sea level fall 3 plant ecosystem in order to better understand one of the most significant faunal events during the Tertiary, the dispersal of hipparions into the Old World. Two scenarios can be considered: (1) hipparionines, because of their hypsodont cheek teeth, exploited grassy abrasive vegetation in open landscapes to spread into the Old World or, (2) their dispersal illustrates a new example of the Liem’s paradox This study aims to investigate the feeding ecology of the earliest hipparionines from Europe within a pure CHippotherium primigenium is always dominant in the faunal assemblage. Bernor et al. Hippotherium primigenium” complex.The four localities, Eppelsheim (EP), Höwenegg (HO), Charmoille (CH), and Soblay (SOB), belong to the Late Miocene (MN9–MN10). The first two sites are situated in southern Germany, Charmoille is located in Switzerland and Soblay near Lyon in eastern France. In the latter locality two hipparionine taxa may have co-habited H. primigenium. Stable isotope compositions of tooth enamel combined with dental micro- and mesowear analyses provide complementary information regarding dietary intake, habitat, and niche partitioning. These methods evaluate fundamentally different chemical (isotopes) and mechanical  food properties. Although these dietary proxies are not completely taxon-independent due to some influences of the animals (digestive) physiology and masticatory food processing, this multi-proxy approach allows a refined dietary reconstruction for hypsodont horses. It contributes to a better understanding of the dietary flexibility and will thus help us better understand the successful rapid dispersal of H. primigenium into the Old World. Dental wear analysis will help to resolve the grazer-browser dichotomy while carbon and oxygen isotope analyses will yield complementary information regarding habitat and food properties.Four different dietary proxies are examined: molar mesowear and microwear patterns in combination with carbon and oxygen isotope compositions of enamel carbonate of the early European Hippotherium primigenium , which is presently housed at the Forschungsinstitut Senckenberg (Frankfurt).The Eppelsheim locality (EP) belongs to the oldest deposits of the Miocene Rhine River, exposed at many places in the Rhine-Hesse area of Germany . These f40Ar/39Ar from the hornblende tuff of the Höwenegg sequence Hipparion’ Datum and because it contains completely articulated skeletons of H. primigenium.The Höwenegg locality (HO), located in southern Germany , belongsThe Late Miocene deposits in Soblay  is a sand pit close to the small town of Charmoille in the canton of Jura in northern Switzerland . The fauHippotherium primigenium. Due to a short turnover time of body water  13C) of animal tissues reflects that of the ingested food 3 and C4 plants from dietary intake 3 plants, which include trees, most shrubs and many cold-season, temperate latitudes and high altitude grasses, discriminate strongly against the heavy 13C isotope and therefore have lower δ13C values than C4 plants, ranging from –34 to –22‰, with an average of –27‰±3 4 plants include mostly warm-season grasses that discriminate less against the heavy 13C isotope and therefore have higher, less negative δ13C values of between –17 and –9‰, with an average of –13‰±2 13C by about +14‰ relative to the plants ingested by large non-ruminant herbivorous mammals such as horses, depending on the digestive physiology and rate of methane production of the animal 3 plant diet have average δ13C values of about –13‰ while animals feeding on a pure C4 plant diet have an average δ13C value of about +1‰ 4 grass-dominated grassland ecosystems of savannas known today first evolved globally in the late Miocene 3 plants dominated the vegetation during the Neogene, as indicated by the observed low δ13C values in fossil mammal teeth of herbivores 3 plant-dominated ecosystem C3 grazers and C3 browsers cannot be easily distinguished by carbon isotope analysis of most tissues. However, the enamel carbon isotopic composition in C3 ecosystems enables the niche partitioning and habitat differences of herbivorous mammals to be determined 13C values even within the C3 plant groups, due to variations in light and water availability as well as position in the forest canopy or habitat temperature, depending on latitude and altitude The carbon isotope composition  dietary signal of the physical properties of the last food items consumed by an individual the major factor driving dental abrasion It is worth noting here that two recent studies based on scratch tests and hardness measurements conclude that silica phytoliths do not appear to influence the enamel enough to scratch it, but identify grit as 13C values of the carbonate in the enamel bioapatite from 22 Hippotherium teeth of the four Late Miocene localities EP, CH, HO, and SOB range from −14.6 to −10.2‰  = 14.395; p = 0.002).Average δo −10.2‰ . These vd −11.5‰ , Table 113C variability of all 10 serially sampled premolars and molars from EP, CH and HO is nearly 6‰ (range: −15.2 to −9.5‰), but is small within a single tooth 0.9±0.3‰  = 10.624; p = 0.013). The intra-tooth δ18O variation of all of the 10 serially sampled premolars and molars has a similar total range from –10.0 to –4.3‰  has a much higher variability οφ 4.0‰, which is likely a result of diagenetic alteration in the lower, less mineralized crown part of this unerupted M2 that also gave the highest δ18OCO3 values  have the highest δo –10.2‰ , while t.3 to 2‰ , Fig. 3.13C values of –14.6 to –12.6‰ similar to those of EP and the intra-tooth variation of the δ13C values ranges from 0.6 to 1.4‰. Mean δ18OCO3 values for all teeth has a range from –9.1 to –7.3‰ and the intra-tooth variation is 1.6 to 2.3‰  have low mean δ to 2.3‰ , Fig. 3.13C values have only a narrow range from –11.7 to –11.2‰ and δ18OCO3 values range from –6.4 to –4.8‰. Teeth from SOB have less negative δ13C and δ18OCO3 values than the two EP and CH populations, similar to those of HO teeth.For Soblay only three molars were analyzed in bulk but no serial intra-tooth sampling was performed. δHippotherium primigenium from the four investigated localities were integrated into a model constructed using present day grazing and browsing ungulates through a discriminant analysis (DA). An overall misclassification for extant species is about 11.54% . For microwear analysis, specimens of p = 0.018).H. primigenium, which plots in the central portion of the browser spectrum. In contrast, the SOB sample has the highest abrasion-dominated signature and therefore plots within the range of extant grazing species. The CH sample plots close to the SOB sample with a molar mesowear score of 1.94. The EP population plots within the mixed feeder range sandwiched between the spectrum for browsers and grazers  to high-abrasion dominated mesowear scores . These differences in enamel δ13C values of the investigated H. primigenium teeth thus reflect δ13C variations of the dietary C3 plants (either C3 dicots or C3 monocots) due to seasonal and/or habitat differences. The enamel δ13C values of 60% of the H. primigenium teeth are 1 to 3‰ lower than the mean value of around –11.5‰ expected for herbivores feeding on C3 plants during the Miocene All 13C values ≤ –13‰ for many of the H. primigenium teeth, especially those from EP and CH, suggest feeding was predominantly in forested ecosystems. This is compatible with the palaeoenvironmental reconstructions for these settings based on palaeofloral remains  on the plants eaten. An alternative interpretation would be that the HO and SOB H. primigenium populations fed predominantly on different plants or plant parts  with less negative δ13C values. The HO and SOB specimens also have higher enamel δ18O values  are compatible with browsing on C3 plants in a forested environment. The dental mesowear signature of the EP population supports mixed feeding habits, whereas, the dental mesowear analysis on the CH sample indicates a diet richer in grasses. However, note that such an abrasion-dominated mesowear signal could also be due to high fruit consumption. The hypothesis of grazing traits is challenged by the dental microwear results of these two populations. In fact, only one individual out of 19 from EP and two of 15 from CH can be considered as grazers; the remaining individuals all plot in the browsers group  and short-term  signals do not always corroborate each other and vary from one population to another and even within a single population. Given the different food properties and timescales represented by the various diet proxies this is not surprising. However, the data clearly demonstrate the potential of combined dental wear and stable isotope analysis to obtain refined and complementary dietary reconstructions of herbivores. This multi-proxy approach reveals dietary flexibility and enables us to gain new insights into the palaeoecology of extinct taxa and their feeding behaviour.Altogether the data support opportunistic and mixed feeding habits for H. primigenium populations. For instance, the EP habitat was part of the Rhine River stream system in a flat and well-watered landscape. Water stress thus likely played a much less pronounced role at EP. Similarly, this may have been the case for HO as it was situated near a lake in the Hegau volcanic field on a tuff-dominated bedrock and well-drained limestone bedrock. Given the higher enamel δ13C and δ18O values from HO, and also SOB, compared to EP and CH, the HO and SOB populations may represent open landscape populations, which in turn would be expected to have had a more grass-dominated dietary regime. This is in agreement with the microwear results from HO teeth, but not for those from SOB where there is robust evidence for wet and forested habitat Hippotherium primigenium was thus generally a flexible and opportunistic meal-by-meal mixed feeder highly engaged in browsing, grazing and occasional frugivory and therefore was predominantly a forest dweller during the Late Miocene of Western Europe.Differences in vegetation density and/or water stress may explain some of the dietary differences between the H. primigenium.Despite the correlation of crown height with ecological factors relating to diet in modern herbivorous mammals, the correlation cannot prove that crown height is always a reliable predictor of diet in terms of browsing versus grazing Hipparions’ were widely considered as grazers. However, there is another biological benefit of hypsodonty beyond the adaptation to an abrasive (grass) diet; a greater dietary flexibility. The present data indicate that Hippotherium had a variable C3 plant-based diet ranging from browsing to grazing and was thus a flexible and opportunistic mixed-feeder. At around 11 Ma, when hypsodont hipparions arrived in Eurasia, C4 plants, which comprise the majority of abrasive grasses in extant open environments, were absent or just a minor component in the global flora and did not become widespread until 8 to 6 Ma 4 grasslands during the early Late Miocene (MN9) and could only have met their dietary needs with the C3 environment, which was probably forested. Hippotherium would have predominantly fed on available leafy plants such as trees and shrubs within these forested landscapes of Europe but could also switch to a more abrasive grass or even a fruit-based diet. This ecologic flexibility enabled hypsodont Hippotherium to feed both on abrasive and less abrasive food items, depending on their availability. This was advantageous for survival in environments with changing seasonality as would have occurred following the Mid-Vallesian crisis, with drier and more seasonal climates prevailing in Europe in the Late Miocene Because of their hypsodonty, ‘H. primigenium via Asia due to any potential climatic (arid areas) and geographic barriers being easier to cope with due to its flexible diet. The North American ancestors of the earliest Eurasian hipparions from the Cormohipparion clade are best known from the Early Clarendonian (ca 12 Ma) quarries in Texas and Nebraska Hippotherium primigenium was likely able to graze as well browse, much in the same way as present-day wild asses. This dietary flexibility would explain its rapid dispersal into Eurasia despite climatic and environmental dispersal barriers, and thus was not necessarily linked to the expansion of grasslands for grazing. The hypsodonty of H. primigenium can thus be considered an example of Liem’s paradox in which a specialized phenotype (high crowned cheek teeth) enabled a more flexible mixed-feeding diet.Furthermore, opportunistic mixed-feeding may have facilitated the dispersal of H. primigenium appears to be, its hypsodont dentition does not restrict it to a habitat of abrasive diets. This is also compatible with the reasoning that fallback foods may have more selective force in natural selection than preferred foods H. primigenium to survive in a complex variety of landscapes, which did not necessarily provide the food items that limit hypsodont herbivores to abrasive diets. Hippotherium is thus another example that hypsodonty, although originally an adaptation to abrasive food, can enable a species to become a generalist feeder of both high- and low-abrasion diets, similarly to camelids H. primigenium to expand its habitats and food resources in the increasingly drier and more open Late Miocene landscapes of Europe Given how wide the dietary niche of Hippotherium in Europe as a hypsodont, opportunistic mixed feeder put competitive pressure on the brachydont equid Anchitherium aurelianense which was feeding in the same ecological niche Anchitherium in the Old World following the Mid-Vallesian crisis, with only a short period of coexistence in the upper Vallesian (MN9) H. primigenium and A. aurelianense specimens are thus limited, hampering a direct diet comparison of sympatric individuals. A rapid replacement of Anchitherium by Hippotherium seems quite plausible due to the latter having a high ecological tolerance; because of its hypsodonty it was capable of feeding on abrasive diets. Although some Anchitherium populations in arid areas such as Spain also have an adaptive increase in crown height, those in humid central European areas (i.e. Germany) did not, and they remained brachydont H. primigenium was able to live and feed in a variety of habitats and therefore was a very successful migrant species.The appearance of H. primigenium to occupy a broad dietary niche. Combined dental meso- and microwear together with enamel oxygen and carbon stable isotope analyses on four populations of hipparionines in Europe, amongst the earliest in the Old World, support mixed feeding habits for H. primigenium with a strong reliance on leafy plants rather than on grazing habits. This high-crowned equid was thus adapted to use a wide range of dietary sources and had a large dietary flexibility. Feeding changed partly on a meal-by-meal basis, but was likely also affected by local and seasonal availability of vegetational resources in the natural food supply. This dietary flexibility was likely a key factor for the rapid dispersal of H. primigenium from North America into Eurasia, given the different environmental settings in this region.To better understand the Late Miocene dispersal of the earliest high-crowned hipparionines in the Old World two hypotheses were investigated: (1) hypsodont cheek teeth facilitated the exploitation of grassy abrasive vegetation in evolving open grassland landscapes and, (2) hypsodont cheek teeth enabled H. primigenium, its hypsodont dentition may no longer be regarded as a constraint for forage on abrasive items in open landscapes. Instead, it would rather enable opportunistic feeding from grazing to pure browsing for long periods of time; even when no less abrasive diets were available. The hypsodonty of its cheek teeth would allow H. primigenium to survive in a variety of landscape types which did not necessarily provide the food items that would limit hypsodont herbivores to abrasive diets. Thus Hippotherium is an example of the Liem’s paradox, hypothesizing that specialized phenotypes (here hypsodonty) enable more ecological flexibility in a taxon and the occupation of a broader dietary niche.With the broad feeding habits of H. primigenium might thus be the cause for the rapid replacement of the low-crowned equid Anchitherium aurelianense in Europe during the Late Miocene (MN9–MN10).The arrival of this ecologically flexible equid must have imposed severe competition on brachydont anchitheres that occupied a variable browsing or mixed feeding niche in the Late Miocene of Europe We thank the Forschungsinstitut und Naturmuseum Senckenberg (Frankfurt), the Staatliches Museum für Naturkunde (Karlsruhe), and the Landesmuseum Rheinland-Pfalz (Mainz), the Collections d’Histoire Naturelle de l’Université de Lyon (A. Prieur) and the Naturhistorisches Museum  for access to the fossil specimens investigated in this study. All the specimens were loaned in the aforementioned museum collections and returned after analysis.Hippotherium primigenium from Eppelsheim , Höwenegg , and Charmoille  were analysed for their carbon and oxygen isotopic composition of carbonate in the apatite  were also analysed for their carbon and oxygen isotope composition of carbonate in the apatite  and oxygen (δ18OCO3) isotopic composition of the carbonate in the apatite. About 2 mg pretreated enamel powder was reacted with 100% H3PO4 for 90 minutes at 70°C using a ThermoFinnigan Gasbench II 2 were measured in continuous flow mode on a Finnigan Delta Plus XL isotope ratio gas mass spectrometer at the University of Lausanne and Tübingen. For this reaction the same acid fractionation factor as between calcite and CO2, was assumed to be applicable. The measured carbon and oxygen isotopic compositions were normalized to the in-house Carrara marble calcite standard that has been calibrated against the international NBS-19 calcite standard. The isotope composition of tooth enamel apatite is reported in the usual δ-notation in per mil (‰) relative to the known isotope reference standard VPDB with δ13C or δ18O (‰) = [(Rsample/Rstandard) −1]×1000, where Rsample and Rstandard are the 13C/12C and 18O/16O ratios in the sample and standard, respectively. Precision for the carbon (δ13C) and oxygen (δ18O) isotopic composition of carbonate in the apatite is better than ±0.1‰ and ±0.15‰, respectively. The NBS 120c Florida phosphate rock standard, also pre-treated after Koch et al. 13CVPDB = –6.23±0.09‰ and δ18OVPDB = –2.23±0.10‰ (n = 9).The enamel powder was pretreated according to Koch et al. After initial examination, many specimens were excluded from analysis because they were physically altered during transportation or compaction of sediments Regarding dental microwear analysis, several protocols were developed to quantify occlusal wear patterns at a microscopic scale. Differences concern casting procedures applied and the data acquisition. In this study the protocol of Merceron et al. Dental facet development on the molar surfaces of herbivorous ungulates appears to be strongly tied to feeding styles Figure S113C and δ18O values of enamel and dentin H. primigenium teeth from the Höwenegg locality as well as on sediment sample from the main fossil-bearing layer.δ Note the large difference in δ13C values indicating a diagenetic alteration of the dentine while enamel still has values typical for C3 feeders.(TIF)Click here for additional data file.Table S1C and O isotopic compositions of the serial sampled Eppelsheim H. primigenium teeth.(XLS)Click here for additional data file.Table S2C and O isotopic compositions of the Höwenegg H. primigenium teeth.(XLS)Click here for additional data file.Table S3C and O isotopic compositions of the Charmoille H. primigenium teeth.(XLS)Click here for additional data file.Table S4Summary statistics of the main dental microwear variables for the browsing and grazing ungulates that composed the comparative dataset.(DOC)Click here for additional data file.Table S5List of extant species used for mesowear analysis.(DOC)Click here for additional data file.Text S1Detailed site description of the fossil localites.(DOC)Click here for additional data file.


Both diets were tested at two water oxygen levels: hypoxia vs. normoxia. Trout consumed 29% less food under hypoxia compared to normoxia (p<0.001). Under both hypoxia and normoxia trout significantly reduced food intake by 11% and 16% respectively when fed the imbalanced compared to the balanced amino acid diet. Oxygen consumption of the trout per unit body mass remained identical for both diet groups not only under hypoxia but also under normoxia (p>0.05). This difference in food intake between diets under normoxia together with the identical oxygen consumption supports the hypothesis that food intake in fish can be constrained by a set-point value of oxygen consumption, as seen here on a six-week time scale.Compromisation of food intake when confronted with diets deficient in essential amino acids is a common response of fish and other animals, but the underlying physiological factors are poorly understood. We hypothesize that oxygen consumption of fish is a possible physiological factor constraining food intake. To verify, we assessed the food intake and oxygen consumption of rainbow trout fed to satiation with diets which differed in essential amino acid (methionine and lysine) compositions: a  Both diets were tested at two different water dissolved oxygen (DO) concentrations (hypoxia vs. normoxia). The conceptual illustration of the hypothesis tested is shown in Therefore, we hypothesize that oxygen consumption is one of the possible physiological factors (constraints) which can limit food intake in fish. We propose that even at normoxic conditions and in the absence of other potential constraints on food intake, food intake in fish can be constrained by a set-point value of oxygen consumption . In the present study, we assessed the food intake and oxygen consumption of rainbow trout fed diets, which were contrasting in their composition of two essential amino acids . Trout fed the amino acid balanced diet had better growth than trout fed the amino acid imbalanced diet. Likewise, trout kept under normoxia had a larger final body weight and higher growth compared to fish kept under hypoxia , being 45% higher in trout fed the imbalanced than in those fed the balanced amino acid diet. The protein retention efficiency was thus significantly lower (24%) in trout fed the imbalanced than in those fed the balanced amino acid diet. Both nitrogen excretion and retention efficiency were unaffected by the water DO level and its interaction with diet  and imbalance amino acid (142.6 mg kg−0.8 h−1) diet (p = 0.36). Despite the differences in food intake under normoxic conditions, the oxygen consumption of trout fed the balanced (171.9 mg kg−0.8 h−1) or the imbalanced amino acid (169.2 mg kg−0.8 h−1) diet were equal (p = 0.36). There was no significant interaction effect between water DO level and diet on the oxygen consumption of trout (p = 0.31).The oxygen consumption of trout was lower under hypoxia than normoxia, which was due to the applied contrast in water DO levels b. Undervs. normoxia).We found that irrespective of the water DO levels, the rainbow trout fed the amino acid imbalanced diet did not increase food intake to compensate for the inadequate amount of lysine and methionine supplied by this diet. Growth was reduced in trout fed the amino acid imbalanced diet, in agreement with data from most of the amino acid requirement studies in fish 2 per g DPI, which was about 18% higher than when fed the amino acid balanced diet (841 mg O2 per g DPI). The higher oxygen consumption in the amino acid imbalanced diet groups is likely to be attributed to an obligatory increase in amino acid deamination and the further processing of amino acid carbon skeleton towards de novo lipogenesisRegulation of food intake in fish is influenced by various physiological conditions, which interact with dietary and environmental factors Of interest, exactly similar observations were made under normoxia as under hypoxia. Even at non-limiting water DO level, rainbow trout reduced their food intake when fed the imbalanced amino acid diet compared to the balanced diet. Moreover, even under normoxia, trout of both dietary groups displayed identical oxygen consumption, although the oxygen consumption was higher than those seen under hypoxia. Unlike under hypoxia, this diet-specific reduction in food intake points toward a constraint imposed by factors other than by the availability of water DO . As mentioned above, the metabolic handling of the amino acid imbalanced diet under normoxia also requires higher oxygen consumption per unit of digested protein intake compared to the balanced diet. As such, the equal oxygen consumption of trout in both the dietary groups together with the differences in voluntary food intake  suggestsThis study complied with the Dutch law on experimental animals and was approved by the ethical committee for animal experiments of Wageningen University (DEC: 2011016.d).Oncorhynchus mykiss; Mohnen Aquaculture, Germany) with an average initial body weight of 52.5 g±1.2 g (mean±SD) were randomly allocated among 12 tanks (30 fish tank−1) of the aquatic metabolism unit . The tanks were connected to a closed recirculation system, equipped to adjust and monitor water flow and oxygen concentration in the inflow water of each tank of the fish housing facility, as for details reported elsewhere −1), nitrite (<0.08 mg N l−1), nitrate (<32 mg N l−1), total-NH3-N (<0.15 mg N l−1).Three hundred and sixty juvenile rainbow trout  amino acid diet and ‘low’ with a balanced (adequate) amino acid diet. Thus, two diets differing in the limiting amino acid content (i.e. lysine and methionine) were prepared: a balanced amino acid diet which meet the lysine (5.1 g per 16 g N) and methionine (2.4 g per 16 g N) requirement for rainbow trout Treatments were designed in a 2 by 2 factorial setup with dietary amino acid composition and water dissolved oxygen (DO) levels as main factors, each consisting of two levels; an amino acid ‘balanced’ −1 with mean water DO level of 10.2±0.2 mg l−1, and the mean DO content in the outflowing water remained above 8.0 mg l−1. When necessary, pure oxygen was injected into the inflow water in order to maintain outflow DO content above 8.0 mg l−1. The hypoxia condition was created by reducing the rate of water inflow (1.94±0.02 l min−1) with a mean DO content of 9.8±0.2 mg l−1, and the mean DO content in the outflowing water remained below 6.0 mg l−1 and above 4 mg l−1. The level of DO content for the hypoxia treatment was decided based on the reported incipient DO level of about 6.0 mg l−1 for rainbow trout The difference in dissolved oxygen (DO) level in the water was created by adjusting the rate of water flow into the tanks. The water volume was kept constant at 200 l in all tanks. For the normoxia groups, the rate of water inflow into tank (mean±SD) was kept at 7.9±0.03 l min−0.8 h−1) −0.8 d−1) −1, 2-phenoxy ethanol) at the start and end of the experiment 36 h after the last meal. Fish used to determine initial (15 fish) and final body composition (8 fish tank−1), and the remaining fish at the end of experiment were euthanized by an overdose of anaesthesia . Fish samples for body composition were stored at −20°C until further analysis.The treatments were randomly assigned among 12 tanks to have triplicates for each treatment group. During the experimental period of 6 weeks, fish were hand-fed with their respective diets twice daily to apparent satiation for an hour (09.00–10.00 and 16.00–17.00 hrs). At each feeding session, feed given and uneaten feed were recorded; in addition, uneaten pellets were collected and counted to determine the food intake accurately. Faeces were collected to determine nutrient digestibility in a similar way as described earlier Whole fish samples were pooled per tank, ground, and subsequently freeze-dried before analysis. Analyses of fish were done in triplicates for dry matter (105°C for 24 h), crude protein , crude fat  and energy  as described previously p<0.05). Normal distributions of the residuals were verified using Kolmogorov-Smirnov test (PROC UNIVARIATE).All parameters of fish growth performance, food intake and body composition were calculated as per formulae mentioned earlier


In the present study we investigate the effects of functional BK channel deletion on cognition using a genetic mouse model with a knock-out of the gene for the pore forming α-subunit of the channel. We tested the F1 generation of a hybrid SV129/C57BL6 mouse line in which the We first evaluated hearing and motor function to establish the suitability of this model for cognitive testing. Auditory brain stem responses to click stimuli showed no threshold differences between knockout mice and their wild-type littermates. Despite of muscular tremor, reduced grip force, and impaired gait, knockout mice exhibited normal locomotion. These findings allowed for testing of sensorimotor gating using the acoustic startle reflex, as well as of working memory, spatial learning and memory in the Y-maze and the Morris water maze, respectively.Prepulse inhibition on the first day of testing was normal, but the knockout mice did not improve over the days of testing as their wild-type littermates did. Spontaneous alternation in the y-maze was normal as well, suggesting that the BK channel knock-out does not impair working memory. In the Morris water maze knock-out mice showed significantly slower acquisition of the task, but normal memory once the task was learned. Thus, we propose a crucial role of the BK channels in learning, but not in memory storage or recollection. The large-conductance, voltage- and calcium-activated potassium channels (BK channels) are expressed throughout the mammalian nervous system Over the last several years evidence has accumulated that alterations in the expression and function of BK channels are involved in a number of mental disorders. So far, functional alterations of these channels have been linked to mental retardation In order to explore the role of BK channels in cognitive processes we tested a mouse model lacking functional BK channels. Previous studies found that BK channel knock-out mice with a 129SVJ inbred background have impaired hearing After confirming normal hearing and locomotion, we first tested sensorimotor gating. Sensorimotor gating is a pre-attentive process that filters sensory information for salient stimuli within the environmental noise. In all patient groups linked with an alteration in BK channel expression disruptions in sensorimotor gating have been described , and 19 homozygote knock-out mice , but different, randomly selected, subsets of these animals were used for the single tests. The hearing test was performed on a separate set of animals (for details see respective method sections). The animals were litter- and/or age matched. We only tested mice at ages from 3 to 7 months to avoid effects of aging.We used mice of the F1 generation of a hybrid SV129/C57BL6 line with deficient BK channel function. BK channel function was abolished by deleting the All mice were generated and genotyped at the Pharmaceutical Institute, University of Tübingen, Germany. In order to ensure proper tracking each mouse got an individual ear-tag when collecting the DNA sample for the genotyping. The mice were shipped to Canada at the age of 1.5-3 months and subsequently allowed to acclimate for 2 weeks before behavioural testing started. Mice were group housed with mixed genetic background within groups, with a 12 hour light-dark cycle and with ad libitum food and water. Testing only occurred during the light cycle from 7am to 7pm. If animals were tested repeatedly over several days, the time of testing for each animal was kept constant.All procedures were in accordance with the ethical guidelines of the Canadian Council on Animal Care (CCAC) and approved by the University of Western Ontario Animal Use Subcommittee.Hearing measurements were conducted at the University of Tübingen, Germany, and were in accordance with institutional and national guidelines following approval by the University of Tübingen, Veterinary Care Unit, and the Animal Care and Ethics Committee of the regional board of the Federal State Government of Baden-Württemberg, Germany.−/− mice (6/2), as described previously Auditory brainstem responses (ABR) were recorded from 10 WT  and 8 BKα+/−: 10/6, BKα−/−: 9/7) was measured in 5 trials per animal. The animals were between 7 and 9 months (34±2 weeks) old at the time of testing. A force gauge  was connected to a metal grid which allowed the animal to hold on. The animal was held by the base of its tail and lowered towards the grid until it gripped it with both front limbs. Then the animal was gently pulled back until it lost the grip. Between trials the animals were allowed to recover in their home cages for about 10 minutes. For subsequent analyses the results from the 5 trials were averaged for each animal.In order to assess muscular strength, the grip force test was used. The front limb strength of 16 animals of each genotype , and 10 BKα−/− mice (7/3) aged 7 to 9 month (33±2 weeks) were tested on the CatWalk . Animals traversed a transparent walkway through which they were video-taped. Animals were allowed to walk back and forth until a minimum of 5 consecutive steps could be recorded. Using the CatWalk™software  the paw prints of the animals were tracked. We analyzed stride length and paw print area of each individual paw, as well as inter-limb coordination using the regularity index (RI) as introduced by Hamers et al. To evaluate gait 11 WT , 11 BKα+/−: 10/6, BKα−/−: 9/7) at the age of 6 to 9 month (30±2.5 weeks). Each animal was placed in a square open field box  for 2 hours. During the whole time of testing the room was moderately lit (200 lux). Using the VersMax™ software (AccuScan Instruments) we analyzed the distance traveled during the 2 hours, the number of rearing movements, as well as the time spent in the center versus the time spent at the margins as a measure of anxiety.General locomotor activity was measured in 16 mice of each genotype , and 19 BKα−/− mice (9/10) were tested as described previously Prepulse inhibition of the acoustic startle reflex was used to measure sensorimotor gating. 18 WT , 17 BKαSound was presented by two loud speakers in each startle box. The first speaker presented a constant background noise (65 dB SPL white noise), whereas the second speaker presented the startle and prepulse stimuli. The sound was calibrated with the sound pressure level measurement package provided by MED Associates (ANL-929A-PC) placing the microphone into the animal holder at approximately the same distance to the speaker as the animals’ head during the test sessions. To record the startle response, the animals were placed into a tube like plastic animal holder which was then mounted on top of a piezo driven transducer which translated vertical movements into a voltage signal. The voltage signal in response to a given stimulus was amplified, digitalized and stored on a computer. We started the recording 50 ms before any stimulus was given and recorded for a total of 500 ms per stimulus.Before the actual testing the animals were acclimatized to the startle boxes for 3 days to reduce anxiety. During these 5 minute long acclimation phases the background noise was presented. On the third day, these 5 minutes were followed by a short input/output (I/O) test to determine the appropriate gain setting for the transducer signal for each individual animal. The I/O test consisted of 12 acoustic stimuli with increasing intensity . Once set, the gain was kept constant for all recordings of a given animal. In order to reduce variability we tested each animal on 5 consecutive days using the following protocol.Animals were first acclimatized to the startle boxes as during the acclimation days, and then habituated to the startle stimulus to avoid habituation effects during the prepulse testing. For habituation, the startle stimulus  was presented 100 times with varying inter-trial intervals (10–20 seconds). The prepulse subsequent test consisted of 7 different trial conditions which were each presented 10 times in a pseudo-randomized order and at varying inter-trial intervals (10–20 seconds). The trial conditions included startle alone trials to determine the baseline startle, and trials with one of two types of acoustic prepulses  leading the startle pulse by 10, 30, or 100 ms. Unfortunately, animals sometimes startled to the prepulse of 85 dB SPL, thus we omitted these trials from further analysis, since effects of muscle fatigue could not be excluded.For each trial the startle amplitude was calculated as the difference between the maximum positive and negative displacement of the transducer induced by the startling animal. Prepulse inhibition was calculated for each animal and each trial condition as PPI (%)  =  (1- average startle amplitude to pulse with prepulse/average startle amplitude to pulse only)*100.+/−: 10/5, BKα−/−: 7/8) aged between 8 and 10 month (36.5±2 weeks) were tested in the Y-maze. The custom-made maze consisted of three white plastic arms separated by a 120° angle. Each arm was 30 cm long, 10 cm wide, and restricted by 20 cm high walls. The room in which the test was performed was moderately lit (200 lux). The animals were placed in the center of the maze and were allowed to explore freely for 5 minutes, during which the number and order of arm entries was recorded. An arm entry was defined as the animal placing all four paws into a given arm. Spontaneous alternation was calculated as the ratio of number of triads  and total arm entries.In order to asses working memory and exploration behavior 15 mice of each genotype  in the Morris water maze. The animals were between 10 and 12 month (46±3 weeks) old at the time of testing. We used a circular pool with a diameter of 1.5 meters and 60 cm high walls. The water was 45 cm deep and at room temperature (24°C). The pool was divided into equally sized quadrants in each of which a squared platform (10×10 cm) could be positioned in the middle. The platform was transparent and about 1.5 cm below the water surface to avoid that the animals can see it. Permanent visual cues on the room walls allowed for spatial orientation. A camera mounted above the pool recorded every trial and animals were tracked using the ANY-maze™ software To assess spatial reference learning and memory we tested 14 mice of each genotype . The platform position was assigned randomly, but was counterbalanced for the genotypes. Once assigned, the platform position for a given animal was not changed between trials and days. However, the starting position of the animal was changed between every trial . If the mouse did not find the platform within 90 seconds it was guided to the platform on the first trial. The animals were allowed to sit on the platform for 10 to 15 second and were then moved back into their home cages. For every trial we analyzed the time it took the animal to reach the platform. On the fifth day we ran a 60 second probe trial without the platform and recorded the time the animals spent in the quadrant where the platform previously was hidden.Data analyses and graphical display were done with Microsoft Excel , Graphpad  and SPSS . Data are expressed as mean ± S.E. We also displayed the standard error in the graphs as measure of precision of the sample mean.Depending on the paradigm, a 2-Way-ANOVA, a MANOVA, or a repeated measurement ANOVA with genotype and gender as between-subjects factors was performed to compare groups.p < .05, **p < .01, *** p < .001.For repeated measurement ANOVAs the Mauchly test was used to judge if the data violated the sphericity assumption. In case of a violation the degrees of freedom were corrected using the Greenhous-Geisser (if ε < .75) or the Huynh-Feldt method (if ε > .75). For post hoc analyses the Sidak t-test was performed. Differences were considered statistically significant when p-values were smaller than 0.05. In the figures significance levels between genotypes were indicated as followed: * −/−), as well as their heterozygous (BKα+/−), and wildtype (WT) littermates for their hearing ability and motor function. Subsequently, we assessed their cognitive functions using a variety of tests.We tested the F1 generation of homozygous BKα knockout mice . Despite this high frequency hearing loss, a progression of hearing thresholds upon aging could not be found within the frequency range of best hearing .Hearing function was measured by ABR thresholds. Thresholds for click evoked ABRs were not statistically different for WT (19.8±5.8 dB SPL) and BKαThus, we concluded that cognitive tests that require normal hearing function can be performed with this animal model, as long as testing occurs in the low and/or mid frequency range.BK channels are abundantly expressed in the cerebellum and other motor areas in the brain. Therefore, deficient BK channel function might affect motor function. Since almost all cognitive tests rely on motor behavior and/or locomotion, we next tested these in our mice.−/− mice had a noticeable muscle tremor, whereas their BKα+/− and WT littermates moved normally. Apart from the just visual evidence, we were able to detect and measure this tremor during the startle testing. A two-way ANOVA revealed that the voltage signal on the movement sensitive platform during the periods before the acoustic stimulation was significantly different for the genotypes . The BKα−/− mice had a significant higher noise (43.0±1.2 mV) compared to the WT  and BKα+/− mice . This increased noise was not due to initiated movements of the mice during this brief period since that would have resulted in much larger signal amplitudes. All the recordings were visually inspected to exclude trials with spontaneous movement. Thus, the increased noise seems to reflect the muscular tremor seen in the BKα−/− mice.All BKαF  =  119.03, p < .001). BKα+/− mice (0.38±0.01 lbf) showed similar strength as WT mice , whereas BKα−/− mice showed significant lower muscular strength compared to both, WT (p < .001) and BKα+/− mice (p < .001). With a grip force of on average 0.17±0.01 lbf they had less than half the muscle strength of their WT littermates. There was no gender effect, nor a genotype x gender interaction for the grip force.In order to assess the muscular strength of the mice we used the grip force test . A two-w−/− mice. A MANOVA on all parameters captured from the catwalk, reported a highly significant difference between genotypes   =  5.00, p < .001, no gender effect and no genotype x gender interaction).In order to evaluate the ability of the mice to walk properly, they were tested on the cat walk. We analyzed stride length and paw print area of each individual paw, as well as inter-limb coordination. An example of paw prints and the respective timing of the paws is given in F  =  32.6, p < .001). The stride length of the BKα−/− mice was about 30% shorter than that of WT and BKα+/− mice, with an average stride length of 5407±150 mm as opposed to 7815±220 mm (p < .001) and 8219±330 mm (p < 0.001), respectively. In contrast, the paw print area did not differ between genotypes for any of the paws (Most prominent was the difference between the genotypes in the stride length . The effthe paws .+/− mice and only 27% (3/11) of BKα−/− mice were rated perfect. Indeed, the average regularity index for the genotypes differed significantly   =  4.35, p  =  .023, BKα-/-  =  91.5±2.2% the BKα−/− mice showed significantly less regularity in their walking pattern than the WT mice which had an average RIWT  =  99.3±0.5% (p  =  .037). With a RI  =  95.84±2.2% the BKα+/− mice showed intermediate inter-limb coordination, but it was not significantly different to either the WT (p  =  .416), or the BKα−/− mice (p  =  .507).In order to grade the inter-limb coordination, the regularity index (RI) was calculated. Whereas 81% (9/11) of the WT animals were rated as having a perfect RI, only 45% (5/11) of the BKα−/− mice showing tremor, reduced muscle strength, and gait disturbances, they exhibited normal locomotor behavior   =  0.64, p  =  .801, no gender effect, nor genotype x gender interaction). On average the BKα−/− mice traveled the same distance (989±127 cm) with the same speed (9.8±0.7 cm/s) and showed similar rearing activity (30.1±8.8) as their WT  and BKα+/− litter mates . There was also no significant difference between genotypes for the respective parameters within the first 5 minutes of the open field test. To further assess locomotion, we also measured the swim speed of the mice in the Morris water maze. We could not detect any differences in swim speed between genotypes   =  1.20, p  =  .314, no gender effect, nor genotype x gender interaction, data not shown).Despite the BKαbehavior . A two h−/− mice show normal locomotion but disrupted fine motor coordination. Therefore, cognitive tests that rely on normal locomotion can be performed with this animal model, as long as fine motor coordination is not required.We conclude that BKαF  =  6.04, p  =  .005, no gender effect, no genotype x gender interaction), but also a significant effect for the day of testing   =  7.198, p < .001) and a day x genotype interaction   =  2.155, p  =  .034). Therefore, we analyzed the first day of testing separately and then additionally analyzed the development of prepulse inhibition across the 5 days of testing.We originally tested prepulse inhibition over five days to reduce variability in the data. Interestingly, the repeated measurements ANOVA  did not only report a genotype effect   =  0.338, p  =  .715), nor an effect of gender, or a genotype x gender interaction   =  5.05, p  =  .010, no gender effect, no genotype x gender interaction). While in the WT mice prepulse inhibition on day five was 34.0±6.1% higher, it was only improved by 5.3±6.7% in the BKα−/− mice.For the first day of testing the repeated measurements ANOVA  on prepulse inhibition did not reveal any significant differences between the genotypes   =  0.04, p  =  .960, −/− mice. However, note that there was a significant difference in the number of total arm entries between the genotypes   =  17.13, p < .001, −/− mice showed twice as many arm entries (22.1±1.8) as the WT  and BKα+/− mice , and therefore spent less time in a arm per entry. It could be observed that they did not explore the arms completely. This possibly indicates a different exploratory behaviour strategy. There was no gender effect, nor a genotype x gender interaction for spontaneous alterations or number of arm entries.We next tested our animals for spatial working memory using the spontaneous alternation task in the Y-maze. A two-way ANOVA reported no significant differences between the genotypes   =  11.33, p < .001, no gender effect, nor genotype x gender interaction). The ANOVA also found a main effect for the day   =  57.65, p < .001) and for the trial   =  5.306, p  =  .002), but there was no interaction with genotype for neither of them, nor a day x trial x genotype interaction. The BKα−/− mice took longer to reach the platform throughout all trials .Finally, we tested the animals for spatial reference learning and memory in the Morris water maze. The animals underwent four training days with four trials a day. As mentioned previously, there was no difference in the swim speed for the genotypes. However, repeated measurements ANOVA  for the time to reach the platform reported significant differences between genotypes  compared to the WT (τ  =  0.80 days) and BKα+/− mice data   =  2.17, p  =  .057), nor a gender effect, or a genotype x gender interaction. The WT and BKα−/− mice spent an equal amount of time in the quadrant where previously the platform was hidden (51.6±4.2% and 56.2±3.9% respectively). Thus, we conclude that the BKα−/− mice do not have spatial memory deficits.Once the task was learned, the BKαuadrants . A MANOVIn summary, the present study suggests that a knockout of the α-subunit of the BK channel results in deficits in learning. The spatial working memory and the spatial reference memory seem not to be affected.In the present study we used for the first time a genetic model of BK channel deficiency to test the role of BK channels in cognition. We used mice with a hybrid SV129/C57BL6 background which are not hearing impaired and show normal locomotion. We found that the ablation of functional BK channels disrupts prepulse inhibition learning and slows down the acquisition of new tasks, but does not affect memory.−/− mice could perform all tasks required for the cognitive tests that we employed. One of the major concerns was their hearing. Rüttiger et al. We first verified that our hybrid SV129/C57BL6 BKαIn the present study we used a hybrid SV129/C57BL6 mouse line and we could not find any age depending reduction of hearing thresholds for the click stimuli, which were later used in the acoustic startle reflex experiments. However, we still see an effect of the BK channel function loss on the high frequency range hearing thresholds. This high frequency hearing loss should not affect the acoustic startle reflex experiments since the click stimuli used there only include low to mid range frequencies. BK channels can not only alter hearing thresholds, but also temporal coding of sounds Another major concern with general BK channel knockout mice is their motor function. BK channels are abundantly expressed in the cerebellum BK channels deficiency has been implicated in mental retardation/ fragile X syndrome Interestingly, we found a deficit in prepulse inhibition learning. Wild-type mice showed an increase in prepulse inhibition across the five days of testing to an average of around 60% whereas the knockout mice did not substantially improve. This effect cannot be attributed to differences in the baseline startle, since both wild-type and knockout mice had similar baseline startle amplitudes. A floor effect in the knockout mice can be further excluded because the absolute startle amplitude in the wild-type mice was smaller than in the knockout mice in the presence of a prepulse, and all startle amplitudes were at least twice the noise level and well detectable (data not shown). Experience-dependent increase in prepulse inhibition has been described before and has been attributed to an associative learning process which links the prepulse with the startle stimulus in a timely manner Another common symptom of the mental disorders linked with BK channel deficiency is cognitive disruption. BK channels are expressed almost throughout the entire central nervous system, but the expression is particularly high in the hippocampus in vitro whole-cell recordings in vivo spontaneous firing rate of hippocampal CA1 How exactly the BK channels are involved in learning is not fully understood yet. BK channels mediate the fast after-hyperpolarization and thus regulate intrinsic neuronal excitability Interestingly, memory, once it is established seems not to be affected by the BK channel knock-out. Both, working memory , as well as the spatial reference memory (which was measured on the probe day in the Morris water maze), were comparable between the knock-out and the wild-type mice.The present study shows that BK channel knock-out mice do not improve prepulse inhibition of startle over days and are slower in learning the Morris water maze task, suggesting a critical role for the BK channels in mechanisms underlying associative learning. BK channels may therefore become an increasingly interesting target for the development of new drugs for enhancing cognitive function.Figure S1Weight gain in males (blue) versus females (red) and wild-types (dots) versus knock-out (squares) mice. No difference in body weight gain was observed between any of these groups during 4 to 15 weeks of age.(TIF)Click here for additional data file.Figure S2ABR hearing thresholds for WT (black circles) and BKα-/- mice (grey squares) for click stimuli did not change significantly with age. Animals measured on more than one time point are connected with lines.(TIF)Click here for additional data file.


Bathymodiolus thermophilus. Eight allozyme loci and DNA sequences of four nuclear genes were added to previously published sequences of the cytochrome c oxidase subunit I gene. Our data confirm the presence of two barriers to gene flow, one located at the Easter Microplate (between 21°33′S and 31°S) recently described as a hybrid zone, and the second positioned between 7°25′S and 14°S with each affecting different loci. Coalescence analysis indicates a single vicariant event at the origin of divergence between clades for all nuclear loci, although the clines are now spatially discordant. We thus hypothesize that the Easter Microplate barrier has recently been relaxed after a long period of isolation and that some genetic clines have escaped the barrier and moved northward where they have subsequently been trapped by a reinforcing barrier to gene flow between 7°25′S and 14°S.Comparative phylogeography of deep-sea hydrothermal vent species has uncovered several genetic breaks between populations inhabiting northern and southern latitudes of the East Pacific Rise. However, the geographic width and position of genetic clines are variable among species. In this report, we further characterize the position and strength of barriers to gene flow between populations of the deep-sea vent mussel  Genetic structure is easier to detect and understand in a one-dimensional system than in a two-dimension space When detecting a genetic cline, one should consider the possibility that the location of this cline may be due to the presence of a natural barrier to dispersal because clines are expected to be trapped by such a barrier The geographic region in which a balance between dispersal and selection is maintained is defined as a tension zone. Tension zones tend to stabilize over natural barriers to dispersal Deep-sea hydrothermal vents are patchily distributed along mid-ocean ridges and back-arc basins. Along the East Pacific Rise (EPR), they follow a one-dimension pattern, ideal for testing an isolation-by-distance model of populations Alvinella pompejana displayed an abrupt separation of the populations across the equator Bathymodiolus thermophilus exhibited a smooth clinal distribution of mitochondrial lineages along the EPR (13°N to 21°33′S) B. aff. thermophilus further south (31°S–32°S) Comparative phylogeographic analyses of deep-sea hydrothermal vent species have previously shown the presence of a genetic break between the northern and southern regions of the EPR Bathymodiolus species have been supposed to be long-distance dispersers because of their planktotrophic mode of larval development Bathymodiolus have been confirmed in several population genetic studies showing the absence of genetic differentiation across the Atlantic et al.Bathymodiolus units, Audzijonyte & Vrijenhoek et al.B. thermophilus and B. aff. thermophilus (renamed B. antarcticus by the authors). One of the loci, S-Adenosyl Homocysteine Hydrolase (SAHH), displayed a discordant cline position with fixed substitutions around the geographic zone previously identified by Plouviez et al.The likely cause of the clines observed at the EPR has been a source of debate. Although Plouviez In the present study, we obtained allozyme and DNA sequence datasets for four nuclear genes including the SAHH marker and analysed this new dataset together with mitochondrial haplotypes previously obtained from the EPR mussels to further investigate how genetic diversity is structured across the EPR and more specifically to test for the homogeneity of gene divergences across identified barriers. We obtained evidence for the existence of two barriers, including the one positioned at 7°25′S–14°S. We thus propose that a pair of (semi-)permeable barriers along the EPR is likely responsible for the geographically discordant clinal distribution of alleles in this region.No specific permits were required to perform field studies described in this article. No specific permissions were required to access geographic localities and sample specimens . The locations are not privately owned or protected in any way. The field studies did not involve endangered or protected species.Bathymodiolus thermophilus specimens were sampled from seven deep-sea hydrothermal vent fields along the East Pacific Rise (EPR) from 9°50′N to 21°33′S , Mannose phosphate isomerase , Octopine deshydrogenase , Leucine amino peptidase , Glucose phosphate isomerase , Malate deshydrogenase-1 and -2 , and Hexokinase-1 . Alleles were numbered according to their relative mobility from the most frequent allele (labelled as 100) previously determined for the Atlantic species B. azoricus, this species was used as a reference  were estimated for each population along the EPR. The overall genetic differentiation across populations was estimated at all loci using Weir & Cockerham's θ estimator The program Genetix 4.05.2 et al.B. thermophilus were identified from cytochrome oxidase I gene (mtCOI) sequences previously obtained by Plouviez et al.  gene were also designed from the cDNA sequence data . Sequences from Sulfotransferase were sorted in two paralogous genes called Sulfo1 and Sulfo2 from individual allele recaptures, respectively. Because of the low number of sequences recaptured from Sulfo2, only Sulfo1 sequences were used in this study.Genomic DNA was extracted using a CTAB-PVP extraction procedure following Jolly z et al. , Table 1z et al.  were obtet al.et al.et al.et al.Faure http://www.codoncode.com/aligner/). Sequence alignments were initially performed with ClustalW in vitro recombinants and putative artefactual/somatic mutations. Recombinants between different individuals (1–2% of the dataset for each population) from the same PCR set were detected (and removed) based on abnormal combinations of the 5′-tails.DNA sequences obtained from the MR-cloning method were visualized and edited using CodonCode Aligner 2.0.6 (nπ) and Watterson's theta (wθ) were estimated using DnaSP 4.10.3 www.fluxus-engineering.com) B. thermophilus and B. azoricus . This test compares polymorphism and divergence at several loci to detect if at least one of these loci displays a departure from neutral evolution. This test was preferred to the McDonald-Kreitman Neutrality of loci was also tested using a multi-loci Hudson-Kreitman-Aguadé test HKA,  performestφ, estimated with DnaSP 4.10.3 stφ values in the matrix, the EF1α gene and some of the populations for which, at least one gene was missing were discarded from the analysis . An RFLP analysis was then performed using the Hinf I site to detect N/S individuals by screening all individuals from the Bathymodiolus collection. Incubation of PCR-products was done at 37°C for 1.5 hour in a 20 μl total volume containing 17 μl of PCR product, 1X buffer (supplied by the manufacturer) and 10 U of Hinf I (Ozyme™).A B. thermophilus populations located along the EPR. Among the eight allozyme loci, four  were nearly monomorphic with the most frequent allele occurring at a frequency greater than 95% in all populations. The remaining four loci  exhibited enough polymorphism to investigate their allelic distribution over the range of the EPR. Genetic differentiation was very low and not statistically significant overall , showing the absence of any strong genetic structure at allozyme loci along this portion of the EPR. The Mantel Spearman test showed a slight but significant correlation  between the genetic distance (θ/(1-θ)) and the geographic distance between vent fields  population size (NN being slightly higher than SN). However, expansion cannot be confirmed because of the overlap range of Highest Posterior Density among north, south and ancestral populations.The Monmonier analysis identified a barrier between 7°25′S and 14°S for all tested loci  and φst  barrier . An Isol barrier . The estion size , indicatB. thermophilus and B. azoricus across the Isthmus of Panama, divergence times between clades 1 and 2 were estimated at 0.6–0.9 Mya for mtCOI, conforming to geological estimates of the ages of the transform faults in the 7°25′S–14°S area  and using a 8–12 Mya calibration time obtained from the splitting of 4°S area . In cont4°S area . DivergeB. thermophilus and B. azoricus, indicative of balancing or strong purifying selection.The multi-locus HKA test showed a significant departure from neutral evolution (P < 0.02) among genes within clade 1 (no departure among genes within clade 2), indicating that at least one gene could be under selection. Sulfo1 displayed the highest divergence to polymorphism deviation when compared to other genes. The removal of Sulfo1 (possible outlier) from the dataset led the HKA test to refit the model of neutral evolution (P > 0.31). This gene displayed a polymorphism/divergence ratio of 8.66: a value that is more than eight times greater than expected (1.05) with a very low divergence between The RFLP analysis of the SAHH marker revealed that only 13 individuals south of the 7°25′S–14°S barrier displayed at least one nuclear allele corresponding to the northern clade. Except for one individual showing two northern-type alleles in the southern populations, all of these individuals possessed one allele of each clade. These N/S individuals were mainly located at 14°S and decreased abruptly in frequency further south . No N/S Bathymodiolus have high dispersal capabilities B. thermophilus supports this hypothesis in showing a complete lack of differentiation at allozyme markers between the northern EPR  and the Galapagos sites The unpredictable nature of fluid circulation and the high level of fragmentation found at deep-sea hydrothermal vents should result in species with high dispersal capabilities in order to promote long-distance (re)colonization of new vent sites Bathymodiolus thermophilus is impacted by two barriers to gene flow along the EPR: the so-called Easter Microplate barrier .Previous and present works strongly support the hypothesis that the population structure of stφ with geographic distances at the mtCOI locus (significant Mantel Spearman tests) likely reflects the occurrence of a tension zone that originated at the Easter Microplate .The Monmonier analysis and coalescence analyses statistically detected the presence of a barrier to gene flow at 7°25′S–14°S using nuclear and COI genes. Sulfo1 and SAHH loci both showed two sets of divergent alleles distributed in the 9°50′N–7°25′S and 14°S–21°33′S regions, with an almost reciprocal monophyly. The gene encoding Lyso exhibited two sets of divergent alleles that were geographically interspersed over the whole EPR and PAR, indicating that they were able to cross both the Easter Microplate and the 7°25′S–14°S barriers. However, the 1-bp deletion allele found at a high frequency in populations from 14°S-21°33′S was not observed from 9°50′S to 7°25′S nor within individuals from 38°S. The sample size  is far too low to exclude the absence of this deletion at 38°S. The absence of the deletion in the 9°50′N and 7°25′S individuals, however, strongly suggests that the spread of this new deletion has been blocked since its first occurrence in the southern populations (at least across the 7°25′S–14°S barrier). This deletion is however old enough to provide recombinant alleles at a high frequency between the two Lyso divergent clades, as the deletion is present at the tip of both divergent clades see . The geoet al.Mpi, Odh, Mdh 2 and Hk) because of their low level of polymorphism.The barrier was however not sufficient to create an allele frequency shift at allozyme markers, highlighting an apparent discrepancy among marker types . Discrepancies in the genetic differentiation observed with allozyme and DNA markers have regularly been suggested to result from selection at allozyme loci, sometimes claimed to be under balancing selection Geologic and hydrodynamic conditions from the equator to 15°S are consistent with limited gene flow observed for multiple species between north and south EPR Based on tectonic plate history, the Easter Microplate barrier is older than the 7°25′S–14°S barrier. The Easter Microplate originated from the progressive offsetting of the Pacific-Antarctic Ridge (PAR) along with the ‘old’ EPR (Nazca Pacific spreading centre) about 3.88 Mya . This offsetting progressively expanded with the formation of an overlapping spreading center OSC: see . Ridge oet al.et al.et al.st, Contrasting results among genes across the Easter Microplate are in accordance with the presence of a tension zone at the latitude of Easter Island as proposed by Johnson The hypothesis of a relaxation of the barrier at the Easter Microplate is also supported by the geological formation of the microplate itself. If initiated by the offsetting of the PAR and ‘old’ EPR, the plate formation was achieved when the ends of the two expanding ridges joined together following a clockwise rotation with the setting up of insular volcanic arcs (Orongo and Pito rifts) about 2.5–1.75 Mya  were similar to those estimated at the Easter Microplate boundary between the PAC and SEPR clades of mtCOI and EF1α. This convergence in divergence times found at two distinct spatial locations, as well as the fact that nuclear mutation rates should be lower than that of the mtCOI  under differential ecological selection out of four genes is highly unlikely. This therefore suggests that allele frequency shifts are linked to tension zones and are thus able to move. Together with what we know of the geological history of the EPR, the hypothesis of an interchange of clines from the southern barrier to the northern barrier explains the geographic discordance of cline positions.To conclude, we have shown that the clinal distribution of mtCOI haplotypes along the EPR was in fact due to highly dynamic historical/geological processes. A system of two genetic barriers to gene flow, in which one would be progressively relaxed  and the other enforced , can explain the discordant distribution of genetic clines. A volcanic arc joining PAR and EPR probably played an important role in setting up of a secondary contact zone  between individuals across the Easter Microplate. The proposed relaxation/enforcement scenario of barriers is consistent with the occurrence of a biogeographic transition zone along the southern EPR (resulting from the overlap of the North-EPR and the South-Easter Microplate biogeographic provinces) to explain the high diversity of vent fauna species observed between 17°25′S and 21°33′S Table S1GenBank accession numbers of each unique sequence and their geographic distribution. Numbers correspond to the number of individuals having the accession number in a given population. For example, accession numbers KC858658 through KC858662 all have a single individuals recovered in the 9°50′N population.(DOCX)Click here for additional data file.


Parkin gene (PARK2) are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types , we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from Drosophila melanogaster. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function.Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting approximately 1–2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the  Loss of one of these two genes results in mitochondrial pathology and muscle and dopaminergic neuron degeneration. Overexpression of Parkin rescues the phenotypes caused by PINK1 deficiency, but not vice versa, indicating that Parkin intervenes downstream of PINK1Parkin and PINK1 and genes encoding components of the mitochondrial fission/fusion machinery indicate an involvement of the PINK1/Parkin pathway in the regulation of mitochondrial dynamics Studies in Parkin is at steady state essentially cytosolic, and recent work has shown that it selectively and rapidly translocates from the cytosol to depolarized mitochondria with low membrane potential and subsequently induces their autophagic removal in a process called mitophagy Increasing our knowledge about the interactions between Parkin and other cytoplasmic and mitochondrial proteins will provide further biological insights into Parkin function and the intricate relationships between the multiple roles of Parkin. The identification of such Parkin-binding proteins may have a general role in the pathogenesis of PD and elucidate novel therapeutic targets.Drosophila melanogaster and genome-wide association studies (GWAS) in humans. Our study identified novel candidate Parkin-binding proteins for involvement in cell death processes, protein folding and response to unfolded protein, the fission/fusion machinery, and the mitophagy pathway, and the combined results of the bioinformatics analyses were used to prioritize them into different selection levels.In this study, we report a comprehensive set of novel candidate Parkin-binding proteins identified by Tandem Affinity Purification (TAP)/mass spectrometry (MS) interaction screens. Following the established “guilt by association” strategy, where proteins/genes are prioritized if they are found to be related to known disease genes and processes Drosophila and human GWAS. The candidate proteins were prioritized into different selection levels, which were compared to the results of an independent gene prioritization approach. Finally, two candidates were tested for interaction to Parkin by co-immunoprecipitation.Protein-protein interaction data for the candidate Parkin-binding proteins obtained from the TAP experiments and the proteins known to cause heritable forms of parkinsonism were derived from public databases, and the respective biological processes and pathways were analyzed and compared. Network models were applied to investigate the functional relationships between the candidate Parkin-binding proteins and the proteins related to monogenic parkinsonism. In addition, the candidate dataset was compared to results from genetic interaction screens in TAP-tagged Parkin containing protein complexes were purified in a two-stage purification process of protein extracts prepared from whole cell lysates and cytosolic and mitochondrial fractions from HEK293T and SH-SY5Y cells and analyzed by MS. The TAP experiments resulted in different protein datasets listed in In addition, the following datasets were used in the analyses: MonogenicPD, which includes proteins encoded by genes implicated in monogenic forms of parkinsonism The protein interactions of Parkin and MonogenicPD were investigated based on the human interactome network (HNet). We analyzed the interaction network within the RelatedPD dataset, which includes 80 proteins and 206 binary interactions, out of 307 interactions in total. Six of the nine MonogenicPD proteins are Parkin interactors in HNet  to Parkin and to MonogenicPD proteins was computed in SpNet, which is a subnetwork of HNet, including the ParkinTAP candidates, ParkinIP, MonogenicPD, MonogenicPDIP and all proteins in the interconnecting shortest paths between ParkinTAP and MonogenicPD. In total, it includes 4,009 proteins and 290,496 interactions, where most of the interactions  are expanded complexes. The network of the interacting partners of the ParkinTAP candidate protein LRPPRC is visualized in Another example is provided by the network of TOMM70A, which is characterized by only few interactions . It consCalmodulin (CALM1) is one of the candidate Parkin-binding proteins, which interacts with two other MonogenicPD proteins  in HNet. However, CALM1 is a possible artifact, since Parkin was tagged with a calmodulin binding peptide according to the TAP protocol. Therefore, any ParkinTAP candidate that is a calmodulin interactor (96 from HNet) may also be a possible TAP artifact  indicating a high connectivity between the ParkinTAP and MonogenicPD datasets .Parkin-linked parkinsonism, we performed enrichment analyses for the RelatedPD dataset, consisting of ParkinIP and MonogenicPD. As expected, the most significant pathways according to ConsensusPathDB were “Parkinson's disease” (P = 1.6×10−14), “Alpha-synuclein signaling” (P = 1.1×10−08), and “Role of parkin in ubiquitin-proteasomal pathway” (P = 1.5×10−07) (In order to identify the pathways and processes known to be involved in the pathophysiology of PD and in particular 5×10−07) .−19) and “protein ubiquitination” (LEA P = 1.9×10−17). Additional significant processes were related to apoptosis and to mitochondrial and neuronal processes: “cell death” (LEA P = 2.4×10−14), “regulation of cell death” (LEA P = 1.2×10−10), “apoptosis” (LEA P = 6.6×10−10), “mitochondrion organization” (LEA P = 1.4×10−9), “synaptic transmission” (LEA P = 2.8×10−8), “neuron death” (LEA P = 7.7×10−8), and “dopamine transport” (LEA P = 7.8×10−8) (The most significant GO (gene ontology) biological processes were related to Parkin function in the ubiquitin-proteasome system: “protein modification by small protein conjugation” (LEA P = 2.4x10.8×10−8) .−15) and “Processing of Capped Intron-Containing Pre-mRNA” (P = 6.0×10−15). Other enriched pathways relate to protein folding, like “Prefoldin mediated transfer of substrate to CCT/TriC” (P = 2.0×10−10) and “Protein folding” (P = 1.9×10−7), or to protein processing: “Protein processing in endoplasmic reticulum” (P = 1.6×10−8). Pathway enrichment analysis was also performed for the Mito and Cyto datasets separately. In the Mito dataset, the most significant pathways were related to protein folding and oxidative phosphorylation, whereas in the Cyto dataset the most enriched pathways were related to gene expression. Detailed pathway enrichment results, including the ParkinTAP candidate proteins for each enriched pathway, are provided in The most significantly enriched pathways in the dataset of the 203 ParkinTAP candidate proteins were related to gene expression, in particular to RNA processing/splicing and translation: “Ribosome” (P = 2.3×10−21) and “nuclear mRNA splicing, via spliceosome” (LEA P = 1.1×10−19). Several processes related to protein folding and complex assembly were also significantly enriched, like “protein folding” (LEA P = 7.9×10−17), “response to unfolded protein” (LEA P = 1.2×10−7), or “cellular protein complex assembly” (LEA P = 8.8×10−6), as well as mitochondrial processes like “mitochondrial transport” (LEA P = 8.2×10−6). The Mito dataset contained significant terms related to mitochondrial function, which were specific to this dataset: “electron transport chain” (LEA P = 7.2×10−9) and “oxidative phosphorylation” (LEA P = 2.7×10−5). Detailed results are provided in Of the 203 ParkinTAP candidate proteins, 175 were annotated with a GO biological process. The most well represented biological process categories were related to RNA processing and translation, like “translational elongation” (LEA P = 1.3×10−3) (−3), and ParkinTAP candidates annotated to these five processes (or their child processes) are identified in −3) in ParkinTAP: “protein folding”, “cellular macromolecular complex assembly”, and “cellular metabolic process”. ParkinTAP candidate proteins annotated to protein folding and descendant processes are also identified in To identify and prioritize biologically relevant ParkinTAP candidates, we compared the GO biological process enrichment results between ParkinTAP and RelatedPD and identified 19 GO terms that were significantly enriched in both datasets (classic score P≤10−3) . Of thesIn addition, out of the significantly enriched GO terms in RelatedPD, we selected a set of five representative terms (GOSlimPD)  and idenTo further prioritize ParkinTAP candidates, we assessed the functional similarity between the candidate proteins and the proteins included in MonogenicPD. The FunSimPDsub network  represenTo better investigate which ParkinTAP candidates are functionally related to MonogenicPD proteins, we generated the subnetwork FunSimPD_ND1 consisting of only MonogenicPD proteins and the ParkinTAP candidates that are functionally related to them  . FunSimPParkin and PINK1 mutant phenotypes in DrosophilaParkin wing-posture phenotype or caused lethality prior to adult stage. In these cytological regions, 5,420 human orthologues were annotated and an overlap of 94 proteins with our dataset of candidate Parkin-interacting proteins was identified    within a distance of 50 kb of the listed genetic variants. A variant within this gene has been associated with PD in a recent GWA meta-analysis (P = 2.4×10−6) The ParkinTAP candidates were also compared to human GWAS results for PD to look for evidence of potential association signals in or around the genes encoding these candidate proteins. Comparison to the PDGene database A set of criteria was defined to assign a prioritization score (selection level) for each candidate protein, which is listed in the last column of Using Endeavour −3). In addition, seven out of 99 Parkin ubiquitylation targets (class 1 results) are also included in the ParkinTAP candidates from our study. Parkin is again one of the matching candidates, two additional matching candidates have selection level 0 , other four have selection level 3  or selection levels 6 and 7 . The overlap is again statistically significant (P<1×10−3).A recent study provided a systematic identification of Parkin-dependent ubiquitylation targets and interacting proteins For validation of candidate Parkin-binding proteins, we performed co-immunoprecipitation experiments from HEK293T cells for the two candidates shown in Parkin gene was identified 15 years ago Although the In this study we identified 203 candidate Parkin-binding proteins using TAP/MS proteomic screens. The interactions between these proteins were investigated within HNet, and most of them were part of a single connected component. Whereas only three candidate proteins are known Parkin-binding proteins according to HNet  The biological processes enriched in the identified candidate proteins were compared to the processes enriched in the proteins causing monogenic forms of parkinsonism. Several processes, like protein folding, response to unfolded proteins, mitochondrion organization, and cellular protein complex assembly were found to be significantly enriched in both datasets. Also in the pathway analysis, the protein-folding pathway is enriched in the candidate proteins, and protein processing in the endoplasmic reticulum is significantly enriched both in the candidates and in the PD-related proteins.Parkin and PINK1 mutant functions in Drosophila  By analyzing the functional similarity between the candidate proteins and the PD-related proteins, we identified six functional groups related to RNA processing, complex assembly, protein folding, intracellular transport, mitochondrial transport and ATP synthesis, and programmed cell death. These six protein clusters contained 149 candidate Parkin-binding proteins in total. Whereas most of the candidates are functionally similar amongst themselves, in cluster 6 the candidates are all functionally related to the PD-related proteins, with the only exception of YWHAE . A sub-nParkin-linked parkinsonism. Parkin is localized in the endoplasmic reticulum, the Golgi apparatus, the outer nuclear membrane, synaptic vesicles The diverse functions of the Parkin protein partners reported here are consistent with the functional diversity of the pathogenic processes associated with Parkin and PINK1 mutants in DrosophilaParkin expression has been reported to protect cells against multiple forms of stress In vitro derived TAP results contain false positive interactions and do not represent all binding proteins. Although the two sequential purification steps of the TAP method largely reduce the background resulting from non-specific protein binding compared to a single purification step, these contaminants cannot be completely removed. A limitation of the TAP/MS approach, which preferentially detects interactions within a protein complex Furthermore, the purification step involving the Calmodulin-binding peptide has proven to be problematic when many proteins interact with calmodulin in a calcium-dependent manner Drosophila melanogaster provide additional information about the biological relevance of the interaction of candidate proteins and their putative role in genetic pathways related to PD. The utility of combining protein-interaction screening with genetic-interaction screening to validate protein-protein interaction data was shown in a screen for huntingtin-interacting proteins Protein interaction data generated by any method need to be confirmed through experimental validation, like co-immunoprecipitation assays. For one of two candidate proteins tested, we were able to confirm a physical interaction with Parkin. However, whereas immunoprecipitation gives validation of the physical interaction of proteins, genetic screens in model organisms like Prioritization of candidate binding proteins based on functional annotations might result in a “knowledge bias” towards well-characterized genes. In our study, this is partially countered by also considering previously reported protein interactions, which should not be affected by the same type of bias. However, a careful interpretation of the interactome results is necessary given the noise in the public data, in particular regarding complexes, where the exact interaction partners within the complex are unknown In summary, our study has identified novel candidate Parkin-binding proteins with diverse functions that can be associated to the many pathogenic processes of Parkin-linked parkinsonism. The functional diversity of the Parkin-binding proteins and their involvement in cell death processes, protein folding and response to unfolded proteins, the fission/fusion machinery, and the mitophagy pathway further reveals the diversity and complexity of Parkin function and confirms the large impact of Parkin on cellular physiology. Further studies are necessary to generate high quality, comprehensive interaction datasets for other PD proteins, which can be used to identify shared disease pathways and their components. Focusing not just on individual proteins but, on a network of proteins will prove essential to provide new targets for the development of therapeutic interventions.2. To dissipate the mitochondrial membrane potential, cells were treated with the potassium ionophore valinomycin  or the protonophore m-chlorophenylhydrazone (CCCP) .In this study two cell lines were used: Human embryonic kidney cells  and Human neuroblastoma cells . HEK293T were cultured in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin . SH-SY5Y, ATCC CRL-2266 were cultured in DMEM-F:12 (Lonza) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were maintained at 37°C in a saturated atmosphere containing 5% COMitochondria were isolated from HEK293T and SH-SY5Y cells as previously described Parkin cDNA was cloned downstream of the multiple cloning site into the pCTAP expression vector, which encodes two different affinity purification tags . Subsequently, 108 HEK293T and SH-SY5Y cells were transiently transfected with the pCTAP-Parkin vector using the CaPO4 method To identify Parkin-interacting proteins, the InterPlay Mammalian Tandem Affinity Purification (TAP) System was used according to the manufacturer's instructions (Agilent Technologies). TAP was performed for whole cell lysates of HEK293T cells as well as for mitochondrial and cytosolic fractions of treated and not treated HEK293T and SH-SY5Y cells. In brief, full-length Drosophila melanogaster genes were mapped to human orthologous using InParanoid 7.0 Drosophila, which was performed with Ensembl Gene ID. In the following, the HGNC gene symbols are used to identify both genes and their encoded proteins, according to the context.Ensembl Biomart 64 As a source of known protein-protein interactions, iRefIndex 9.0 was used, which combines protein interaction data from multiple primary resources From this network, a shortest path network (SpNet) was derived by selecting all proteins and interactions within the shortest paths between the ParkinTAP candidates identified in our study and the MonogenicPD proteins Pathway enrichment analysis was performed with ConsensusPathDB-human release 23 −3), and the multiple test corrected classic score is used to confirm the significance of the GO terms selected with LEA.Functional annotation was provided by the Gene ontology (GO) project Functional similarity between proteins was computed using FunSimMat release 4.2 as previously described QuickGO Cytoscape 2.8.1 was used for network visualization of protein-protein interactions and functional relationships An independent gene prioritization of the candidate Parkin-binding proteins was performed with Endeavour DC Protein assay (Bio-Rad). The sample was precleared by incubation with protein A Agarose beads (Roche Diagnostics) for 30 min at 4°C. The beads were removed by centrifugation and the samples were incubated overnight at 4° with rabbit anti-Parkin  or control IgG . The antigen-antibody complexes were captured by addition of protein A Agarose for 2 h at 4°C and washed three times with wash buffer . Proteins were released from the beads by heating at 95°C for 5 min in 4x Sample buffer containing DTT (Life Technologies), followed by SDS-PAGE, blotting onto nitrocellulose membranes, and incubation with anti-TOMM70A , LRPPRC , and Parkin  antibodies. Immunoreaction was visualized using the SuperSignal West Dura Chemiluminescence Westernblot Substrate (Thermo Scientific).HEK293T cells were washed with ice-cold PBS, removed with a scraper, and resuspended in lysis buffer . After incubation for 30 min at 4°C, insoluble material was removed by centrifugation at 14,000×g for 10 min. Protein concentrations were determined using the Bio-Rad Figure S1nteraction networks for ParkinTAP candidate proteins CLPX (A), PRKCSH (B), DAP3 (C), and CALU (D). Proteins are represented as nodes and interactions as edges; the edges are drawn as solid and dashed lines for binary and complex interactions, respectively. Interactions to the selected candidate proteins are represented by thicker edges. ParkinTAP ND X are ParkinTAP candidates at network distance X of MonogenicPD, where ParkinTAP ND 1 are direct MonogenicPD interactors.I(TIF)Click here for additional data file.Figure S2Overview of criteria for the definition of the selection levels. The different datasets are labeled according to the legend of (TIF)Click here for additional data file.Table S1Proteins contained in the datasets listed in .(TSV)Click here for additional data file.Table S2ParkinTAP proteins that do not interact with the other candidate proteins.(TSV)Click here for additional data file.Table S3Summary table for all ParkinTAP candidate proteins.(TSV)Click here for additional data file.Table S4ConsensusPathDB enrichment results for the RelatedPD dataset.(TSV)Click here for additional data file.Table S5GO enrichment results for the RelatedPD dataset.(TSV)Click here for additional data file.Table S6ConsensusPathDB enrichment results for ParkinTAP.(TSV)Click here for additional data file.Table S7GO enrichment results for ParkinTAP.(TSV)Click here for additional data file.Table S8GO enrichment results for FunSimPDsub clusters.(PDF)Click here for additional data file.Table S9Gene prioritization results for the ParkinTAP candidates using Endeavour, including the corresponding selection levels according to our prioritization.(TSV)Click here for additional data file.Text S1Legend (DOCX)Click here for additional data file.


We consider a set of N firing rate neurons with discrete time dynamics and a leak term γ. The nonlinearity of the sigmoid is controlled by a parameter g and each neuron has a firing threshold θ, Gaussian distributed (thresholds are uncorrelated). The network is fully connected with correlated Gaussian random synaptic weights, with mean zero and covariance matrix C/N. When synaptic weights are uncorrelated the dynamic mean field theory developed in -3 allows