Contribution of host species and pathogen clade to snake fungal disease hotspots in Europe.

Infectious diseases are influenced by interactions between host and pathogen, and the number of infected hosts is rarely homogenous across the landscape. Areas with elevated pathogen prevalence can maintain a high force of infection and may indicate areas with disease impacts on host populations. However, isolating the ecological processes that result in increases in infection prevalence and intensity remains a challenge. Here we elucidate the contribution of pathogen clade and host species in disease hotspots caused by Ophidiomyces ophidiicola, the pathogen responsible for snake fungal disease, in 21 species of snakes infected with multiple pathogen strains across 10 countries in Europe. We found isolated areas of disease hotspots in a landscape where infections were otherwise low. O. ophidiicola clade had important effects on transmission, and areas with multiple pathogen clades had higher host infection prevalence. Snake species further influenced infection, with most positive detections coming from species within the Natrix genus. Our results suggest that both host and pathogen identity are essential components contributing to increased pathogen prevalence.

Scaling of erythrocyte shape and nucleus size among squamate reptiles: reanalysis points to constrained, proportional rather than adaptive changes.

Small erythrocytes might be beneficial for blood rheology, as they contribute less to blood viscosity than large erythrocytes. We predicted that rheological disadvantages of larger erythrocytes could be alleviated by relatively smaller nucleus size in larger cells allowing higher flexibility and by more elongated shape. Across squamate reptiles, we found that species with larger erythrocytes tend to have smaller ratio of nucleus size to cell size (N : C ratio), but that larger erythrocytes tend to be rounder, not more elongated. Nevertheless, we document that in fact nucleus area changes with erythrocyte area more or less linearly, which is also true for the relationship between cell length and cell width. These linear relationships suggest that nucleus size and cell size, and cell width and cell length, might be constrained to largely proportional mutual changes. The shifts in widely used N : C ratio and elongation ratio (cell length/cell width) with cell size might be misleading, as they do not reflect adaptive or maladaptive changes of erythrocytes, but rather mathematically trivial scaling of the ratios of two variables with a linear relationship with non-zero intercepts. We warn that ratio scaling without analyses of underlying patterns of evolutionary changes can lead to misinterpretation of evolutionary processes.

Frequent infection of urban grass snakes (Natrix natrix) oral cavity with Leptophallus nigrovenosus trematode.

Land disturbances caused by urban development modify and create novel habitats with novel ecological pressures, which in turn may negatively affect remaining wildlife populations, e.g. by altering interspecific interactions. However, it is not clear whether these modified interactions, e.g. parasitism, affect urban wildlife negatively. This is especially true for reptiles, as even parasitism under natural conditions is understudied in this group. We have observed that up to 35% of grass snakes (Natrix natrix) are infected with the trematode Leptophallus nigrovenosus in urban areas of Kraków, while none of snakes sampled in nearby suburban and non-urban forests exhibited this infection. As this trematode typically inhabits the intestine, we suggest that its occurrence in the oral cavity may be a sign of very high infection rates. However, we did not detect any negative effects of observed infection on body size, mass and body condition of affected individuals. On the other hand, competition with other parasite species in suburban and non-urban habitats may be responsible for not detecting L. nigrovenosus in grass snakes from these sites. Nevertheless, interpretation of our findings is difficult, as knowledge on L. nigrovenosus biology is very limited. Our study underlines the necessity to fill the research gap in reptile parasitology.

Climatic conditions and prevalence of melanistic snakes-contrasting effects of warm springs and mild winters.

Climate change is one of the greatest challenges that wildlife is facing. Rapid shifts in climatic conditions may accelerate evolutionary changes in populations as a result of strong selective pressure. Most studies focus on the impact of climatic conditions on phenologies and annual cycles, whereas there are fewer reports of empirical support for climate-driven changes in the phenotypic variability of free-living populations. We investigated whether climatic variables explain the prevalence of colour polymorphism in a population of the grass snake (Natrix natrix) with two morphotypes, the melanistic and non-melanistic ones, in the period 1981-2013. We found that the prevalence of the black phenotype was negatively related to spring temperature and winter harshness, expressed as the number of snow days. According to the thermal melanism hypothesis, a high predation rate during warmer springs may override relaxed thermal benefits and vice versa, i.e. black individuals may perform better than typical ones when thermal conditions in spring are unfavourable. In turn, because they are smaller, melanistic individuals may be exposed to a higher risk of winter mortality, particularly during longer winters. We highlight the need for more studies on the effects of climatic conditions on temporal variation in melanism prevalence in other populations and species as well as in various geographic regions.

Intestinal upregulation and specific dynamic action in snakes - Implications for the 'pay before pumping' hypothesis.

Animals which feed infrequently and on large prey, like many snake species, are characterized by a high magnitude of gut upregulation upon ingesting a meal. The intensity of intestinal upregulation was hypothesized to be proportional to the time and energy required for food processing (Specific-Dynamic-Action; SDA); hence, a positive correlation between the scope of intestinal growth and SDA response can be deduced. Such a correlation would support the so far not well established link between the intestinal and metabolic consequences of digestion. In this study I tested this prediction using an interspecific dataset on snakes gleaned from published sources. I found that SDAduration and SDAscope were positively correlated with post-feeding factorial increase in small intestine mass, but not with microvillar elongation. This indicates that a wide range of whole intestine remodelling (up- but potentially also downregulation) may temporarily prolong meal processing and that a greater magnitude of intestinal growth requires a stronger metabolic elevation. However, these effects do not seem large enough to drive the variation in the entire energetic costs of digestion, because SDAexpenditure was not affected either by intestinal or microvillar growth. I therefore propose that intestinal upregulation elicits non-negligible costs, but that these costs are a fairly small component of the whole SDAexpenditure.

Decline in body size and female fraction in the grass snake (Natrix natrix, Linnaeus 1758) population after 40 years (Southern Poland).

Depletion of free-living populations is often associated with changes in fitness-related traits, e.g., body size. Ongoing decrease in body size has been reported in most vertebrates, but reptiles remain understudied. Moreover, sexual size dimorphism, commonly observed in reptiles, indicates that environmental pressures on body size may appear sex-specific. This can also result in shifts in sex ratio, an aspect even less studied. We investigated body size and sex ratio in population of grass snake (Natrix natrix) surveyed over 40 years ago in comparison with the current state. We found that both sexes express similar magnitude in body size decline. The current sex ratio does not deviate from 1:1, while in the past, females outnumbered males. The observed changes are likely an outcome of several non-mutually exclusive factors. In the studied area, an increase in road traffic and human presence and a drop in prey availability have been documented. Both factors may exert higher pressure on larger individuals, particularly females, due to their high costs of reproduction. It is recorded here that increase in ambient temperatures and summer duration may additionally enhance the mortality risk and resource requirements. Shifts in body size and sex ratio can catalyze further declines in abundance and reproductive potential of the population.

Energy expenses on prey processing are comparable, but paid at a higher metabolic scope and for a longer time in ambush vs active predators: a multispecies study on snakes.

Snakes are characterized by distinct foraging strategies, from ambush to active hunting, which can be predicted to substantially affect the energy budget as a result of differential activity rates and feeding frequencies. Intense foraging activity and continuously upregulated viscera as a result of frequent feeding leads to a higher standard metabolic rate (SMR) in active than in ambush predators. Conversely, the costs of digestion (Specific Dynamic Action-SDA) are expected to be higher in ambush predators following the substantial remodelling of the gut upon ingestion of a meal after a long fasting period. This prediction was tested on an interspecific scale using a large multispecies dataset (> 40 species) obtained from published sources. I found that the metabolic scope and duration of SDA tended to reach higher values in ambush than in active predators, which probably reflects the greater magnitude of postprandial physiological upregulation in the former. In contrast, the SDA energy expenditure appeared to be unrelated to the foraging mode. The costs of visceral activation conceivably are not negligible, but represent a minor part of the total costs of digestion, possibly not large enough to elicit a foraging-mode driven variation in SDA energy expenditure. Non-mutually exclusive is that the higher costs of structural upregulation in ambush predators are balanced by the improved, thus potentially less expensive, functional performance of the more efficient intestines. I finally suggest that ambush predators may be less susceptible than active predators to the metabolic 'meltdown effect' driven by climate change.

Sexual dimorphism and skull size and shape in the highly specialized snake species, Aipysurus eydouxii (Elapidae: Hydrophiinae).

BACKGROUND: Snakes exhibit sexual dimorphism in both head size and shape. Such differences are often attributed to different reproductive roles and feeding habits. We aim to investigate how sexual dimorphism is displayed in the highly specialised fish-egg-eating snake, Aipysurus eydouxii, by analysing two complementary features: body size and skull morphology. METHODS: We used data on body length, weight, and skull shape from 27 measurements of 116 males and females of A. eydouxii. We investigated both sexual dimorphism and allometric (multivariate and bi-variate) properties of skull growth in the analysed data set. RESULTS: We found that although there was female-biased sexual size dimorphism in body length, females were not heavier than males, contrary to what is commonly observed pattern among snakes. Moreover, females tend to possess relatively smaller heads than males. However, we only found very subtle differences in skull shape reflected in nasal width, mandibular fossa, quadrate crest and quadrate length. DISCUSSION: We suggest that the feeding specialisation in A. eydouxii does not allow for an increase in body thickness and the size of the head above a certain threshold. Our results may be interpreted as support for prey-size divergence as a factor driving skull dimorphism since such species in which the sexes do not differ in prey size also shows very subtle or no differences in skull morphology.

Sex-specific growth is mirrored in feeding rate but not moulting frequency in a sexually dimorphic snake.

Sexual size dimorphism (SSD), commonly observed in snakes, may arise from a different growth rate between the sexes. This indicates a sex-specific resource intake that is in fact observable in free-living snakes. It is not so well known whether the sexes can express differential feeding rates under conditions unconstrained by spatial accessibility, competition, etc. Here, I studied sex-specific variation in growth, its correlate-moulting frequency, and feeding rate in a captive group of sexually dimorphic banded water snakes (Nerodia fasciata) with access to food unconstrained by predation, competition or space. I showed that the sexes did indeed differ in relative mass growth in that females grew faster than males (p = 0.02), but such differences were not apparent in the moulting rate (p = 0.19). Such differential growth was mirrored in the sex-specific feeding rate, with females ingesting a larger number of meals than males (p = 0.004). Such variation in feeding rate may be governed by an individual's energy expenditure and can be interpreted as a behavioural tendency that contributes to SSD development, independently of other behavioural characteristics. Sex-specific resource demands may drive the differential effects of increasing resource scarcity on both sexes.

Cloacal Gram-Negative Microbiota in Free-Living Grass Snake Natrix natrix from Poland.

Reptiles appear to be an important vector for Gram-negative pathogens, therefore, they are epidemiologically relevant. However, the composition of reptilian microbiota has been poorly recognized so far. The majority of studies concern exotic reptiles as asymptomatic carriers of Salmonella serovars. Studies of other intestinal bacteria of reptiles are rare. Only recently, the microbiota of free-living European reptiles have been investigated, however, on the basis of small samples, mainly in protected areas. Here, we aim to investigate cloacal Gram-negative microbiota of free-living Natrix natrix. Snakes (N = 45) used in the study were collected in Kraków (Poland) and its vicinity. Nineteen species of Gram-negative bacteria were isolated. The most common species were: Aeromonas hydrophila, Morganella morganii, Proteus vulgaris, Salmonella spp. The bacteria prevalent in N. natrix cloacal swabs are likely to represent the natural intestinal Gram-negative microbiota of the examined snakes. Importantly, the identified bacteria are pathogenic to humans, which clearly highlights the epidemiological potential of free-living N. natrix. The risk of infection is high for immunocompromised humans, children (under 5 years old), elderly persons, and pregnant women. Our study provides the largest dataset on intestinal Gram-negative microbiota of wild snakes. The presence of multiple human pathogens determined by us calls for the necessity of further studies on reptile-transmitted bacteria in anthropogenic environments.

Melanism, body size, and sex ratio in snakes-new data on the grass snake (Natrix natrix) and synthesis.

It is postulated that melanism in ectotherms is adaptive by enhancing thermoregulation, subsequent resource acquisition, and growth. Such effects may differ between the sexes as a result of the differential costs of self-maintenance and reproduction, but empirical support for the sex-specific consequences of melanism remains inconsistent. We studied the effects of melanism on body size and sex ratio in a population of the European grass snake (Natrix natrix) in SE Poland and also carried out a systematic review of the literature on the consequences of melanism in terrestrial snakes. Melanistic grass snakes of both sexes appeared to be smaller than the typical phenotype, which indicates higher predation pressure and minimal thermal benefits for black individuals. A female-biased sex ratio was observed in the typical phenotype, but not in melanistic snakes, suggesting that the costs for females and/or benefits for males are higher in melanistic individuals. In conjunction with earlier studies, our data indicate that the consequences of melanism may be related to the reproductive mode of species. In viviparous species, melanism tends to improve growth and/or body size and is more frequent in females, whereas the opposite holds for oviparous snakes. Further studies on melanism should examine a wider array of species with different reproductive strategies and traits beyond the usual thermal benefits.

Cellular Turnover: A Potential Metabolic Rate-Driven Mechanism to Mitigate Accumulation of DNA Damage.

Oxidative stress, the imbalance of reactive oxygen species and antioxidant capacity, may cause damage to biomolecules pivotal for cellular processes (e.g., DNA). This may impair physiological performance and, therefore, drive life-history variation and aging rate. Because aerobic metabolism is supposed to be the main source of such oxidative risk, the rate of oxygen consumption should be positively associated with the level of damage and/or antioxidants. Empirical support for such relationships remains unclear, and recent considerations suggest even a negative relationship between metabolic rate and oxidative stress. We investigated the relationship between standard metabolic rate (SMR), antioxidants, and damage in blood plasma and erythrocytes for 35 grass snakes (Natrix natrix). Reactive oxygen metabolites (dROMs) and nonenzymatic antioxidants were assessed in plasma, while two measures of DNA damage and the capacity to neutralize H2O2 were measured in erythrocytes. Plasma antioxidants showed no correlation to SMR, and the level of dROMs was positively related to SMR. A negative relationship between antioxidant capacity and SMR was found in erythrocytes, but no association of SMR with either measure of DNA damage was detected. No increase in DNA damage, despite lower antioxidant capacity at high SMR, indicates an upregulation in other defense mechanisms (e.g., damage repair and/or removal). Indeed, we observed a higher frequency of immature red blood cells in individuals with higher SMR, which indicates that highly metabolic individuals had increased erythrocyte turnover, a mechanism of damage removal. Such DNA protection through upregulated cellular turnover might explain the negligible senescence observed in some ectotherm taxa.

Age-Dependent Utilization of Shelters and Habitat in Two Reptile Species with Contrasting Intraspecific Interactions.

Reptiles undergo worldwide decline driven mostly by habitat change. Detailed recognition of factors underlying spatial structure and habitat utilization is therefore a prerequisite of effective conservation of this group. While the body of data on spatial ecology of reptiles is rapidly growing, studies on social factors remain still underrepresented. We studied age-specific patterns of shelter use, range size, and habitat preferences in the context of intraspecific interactions in the smooth snake Coronella austriaca-known to exhibit intraspecific predation-and the limbless lizard slow worm Anguis fragilis-with no such behavior observed. Despite smaller availability of preferred microhabitats, juveniles of smooth snakes occupied habitat and shelters located at the edge of the population range that did not overlap with adults. No such pattern was observed in the slow worm. Our study indicates that intraspecific interactions affect the spatial ecology of squamates. Passive and active protection of habitat must include wide buffers to preserve the poorly detectable young fraction of the population.

More than just the numbers-contrasting response of snake erythrocytes to thermal acclimation.

Acclimation to lower temperatures decreases energy expenditure in ectotherms but increases oxygen consumption in most endotherms, when dropped below thermoneutrality. Such differences should be met by adjustments in oxygen transport through blood. Changes in hematological variables in correspondence to that in metabolic rates are, however, not fully understood, particularly in non-avian reptiles. We investigated the effect of thermal acclimation on a snake model, the grass snakes (Natrix natrix). After 6 months of acclimation to either 18 °C or 32 °C hematocrit, hemoglobin concentration, erythrocyte number, and size were assessed. All variables revealed significantly lower values under warm compared to cold ambient temperature. Our data suggest that non-avian reptiles, similarly as birds, reduce erythrocyte fraction under energy-demanding temperatures. Due to low deformability of nucleated erythrocytes in sauropsids, such reduced fraction may be important in decreasing blood viscosity to optimize blood flow. Novel findings on flexible erythrocyte size provide an important contribution to this optimization process.

High oxidative stress despite low energy metabolism and vice versa: Insights through temperature acclimation in an ectotherm.

Reactive oxygen species (ROS) are inescapable byproducts of energy metabolism and may cause costly damage to biomolecules. Organisms have evolved different means to counter oxidative stress, such as modulation of ROS production, neutralization of produced ROS through free radical scavenging and the repair or removal of the damaged structures. A positive relationship between metabolic rate and ROS production is commonly expected, but the oxidative burden of aerobic metabolism remains poorly understood. We investigated whether acclimation to ambient temperatures imposing variation in standard metabolic rate (SMR) is mirrored in the oxidative status of an ectotherm. Grass snakes (Natrix natrix) acclimated for six months to warm (32 °C) conditions revealed seven times higher SMR compared to cold-acclimated (18 °C) individuals. In contrast to SMR, the amount of damage measured as reactive oxygen metabolites test (dROMs) and abundance of micronucleated erythrocytes was significantly lower in warm-acclimated individuals, while non-enzymatic antioxidant capacity of plasma was unaltered by thermal acclimation. Our results support that high SMR may allow snakes to better cope with oxidative stress, possibly through tissue repair or removal of damaged tissues that also requires energy costs. The reversed association between self-maintenance metabolism and oxidative damage to biomolecules provides novel rational for temperature dependent life histories of ectotherms. How oxidative stress may contribute to the known reduced rates of ectotherm growth or reproduction under cold temperatures or if oxidative stress may even drive such life history trait declines are now important challenges to be addressed.