Carbonic anhydrase IV in lizard chemical signals.

The evolution of chemical signals is subject to environmental constraints. A multicomponent signal may combine semiochemical molecules with supporting compounds able to enhance communication efficacy. Carbonic anhydrases (CAs) are ubiquitous enzymes catalysing the reversible hydration of carbon dioxide, a reaction involved in a variety of physiological processes as it controls the chemical environment of the different tissues or cellular compartments, thus contributing to the overall system homeostasis. CA-IV isoform has been recently identified by mass spectrometry in the femoral gland secretions (FG) of the marine iguana, where it has been hypothesized to contribute to the chemical stability of the signal, by regulating blend pH. Lizards, indeed, use FG to communicate by delivering the waxy secretion on bare substrate, where it is exposed to environmental stressors. Therefore, we expect that some molecules in the mixture may play supporting functions, enhancing the stability of the chemical environment, or even conferring homeostatic properties to the blend. CA-IV may well represent an important candidate to this hypothesized supporting/homeostatic function, and, therefore, we can expect it to be common in FG secretions of other lizard species. To evaluate this prediction and definitely validate CA identity, we analysed FG secretions of eight species of wall lizards (genus Podarcis), combining mass spectrometry, immunoblotting, immunocytochemistry, and transmission electron microscopy. We demonstrate CA-IV to actually occur in the FG of seven out of the eight considered species, providing an immunochemistry validation of mass-spectrometry identifications, and localizing the enzyme within the secretion mass. The predicted structure of the identified CA is compatible with the known enzymatic activity of CA-IV, supporting the hypothesis that CA play a signal homeostasis function and opening to new perspective about the role of proteins in vertebrate chemical communication.

Combined Effects of Clime, Vegetation, Human-Related Land Use and Livestock on the Distribution of the Three Indigenous Species of Gazelle in Eritrea.

The status and habitat selection of the three species of gazelle indigenous to Eritrea, i.e., Nanger soemmerringii, Gazella dorcas and Eudorcas tilonura, are not well known. In this study, we analyzed the present distribution of the three species in the country in order to identify preferred habitats and assess the effect of human disturbance (land use for agricultural purposes and livestock) on species occurrence. These data represent baseline information for evidence-based strategies for conservation of the three species in Eritrea. Presence/absence data of the three species in each of the 67 administrative subregions (Sub Zoba) composing the country were collected using direct (field surveys) and indirect methods (questionnaires). For each sampling unit, we collected fifteen environmental variables, of which three are associated with climatic features, eight with vegetation structure and four with human disturbance (human-related land use and livestock). The occurrence probability of each species was modeled through Generalized Linear Models (GLM). The analyses showed that Dorcas gazelle occurred more frequently in warmer conditions and in a wide range of natural vegetation types. Heuglin's gazelle occurred in warmer regions with higher seasonality in both temperature and precipitation with a preference for closed woody and open grassland areas. In the case of Soemmerring's gazelle, the GLM with climatic variables predicted a preference for warmer conditions but with lower seasonality of temperature and precipitation. The species also seemed to prefer arid and semi-arid open vegetation. Human disturbance is the variable with the strongest, negative, effect on the species occurrence. Indeed, the occurrence probability of each species decreased with increasing livestock density and agricultural land use. Most of these gazelle occurred in unprotected areas, thus the human-related activities are undoubtedly the most important threat for the three species of gazelle in Eritrea. Therefore, the establishment of protected areas that preserve the potential optimal habitats for gazelle and reduce the impact of livestock ranching are essential to ensure a future for these gazelle in Eritrea.

Species-Specific Spatial Patterns of Variation in Sexual Dimorphism by Two Lizards Settled in the Same Geographic Context.

The evolution of sexual dimorphism (SD) results from intricate interactions between sexual and natural selections. Sexually selected traits are expected to depend on individual condition, while natural selected traits should not be. Islands offer an ideal context to test how these drivers interact with one another, as the size is a reliable proxy for resource availability. Here, we analysed SD in body size (snout-vent length) and head shape (assessed by geometric morphometric) in two species of lizards (Podarcis muralis and P. siculus) inhabiting the Tuscan archipelago (Central Italy). We found a strong SD variation among islands in both species. Furthermore, in P. muralis emerged some significant correlations between SD and island size, supporting the occurrence of possible effects of individual condition on SD. By contrast, SD in P. siculus followed opposite trajectories than in P. muralis, suggesting that in this species, natural selection could play a major role as a driver of SD. Our findings show that natural and sexual selection can interact in complex ways, and the responses are species-specific. Therefore, spatial patterns of variation in SD may strongly differ among species, even when they settle in the same geographic contest.

Haemosporidian infections in wild populations of Podarcis muralis from the Italian Peninsula.

Parasites can significantly influence the ecology, behaviour and physiology of their hosts sometimes with remarkable effects on their survivorship. However, endemic parasites or those not associated with obvious clinical disease have been partly neglected in the past decades comparatively to the most pathogenic ones. Apicomplexa are an important example of blood parasites that have been broadly investigated, although it can be difficult to determine the effects of infections at the population level, especially in widespread species. Such is the case of the common wall lizard (Podarcis muralis). We investigated 61 populations across Italy between 2008 and 2017 and recorded snout­vent length, latitude, date of collection and took blood samples for parasite count. We modelled parasite prevalence and load in a Bayesian framework. Parasites were present in all populations but 1 and in 13 of them all individuals were parasitized. We recorded almost identical responses for probability of infection and parasite load in both sexes, directly proportional to body size and inversely proportional to latitude, with a peak in cooler months. Therefore, haemosporidians can be very common in P. muralis, although their presence can vary significantly. Moreover, such a high prevalence makes it necessary to investigate to what extent haemosporidians affect hosts' survivorship, taking into consideration abiotic and biotic factors such as temperature, hormone levels and immune response.

Patterns of variations in dorsal colouration of the Italian wall lizard Podarcis siculus.

Research on animal colouration has grown exponentially in the last decade thanks to multidisciplinary approaches. Most studies are focused on trade-offs between communication and mimicry, which represent the two main constraints and drivers of the evolution of body colourations. Reptiles are excellent model species for investigating this field of study and lizards in particular show great variability of body colourations and their functions. We studied the lizard Podarcis siculus, analysing the variations of dorsal colour of three populations and obtained clear patterns of seasonal and ontogenetical variation of dorsal colour. According to baseline colour, males were greener and brighter than females, although no difference in saturation was recorded. According to seasonal variations, analyses showed that both sexes significantly vary in colour over the year: males reached higher peaks of hue and saturation later than females during spring, while females showed higher peaks of brightness and reached earlier similarly to hue and saturation. Ontogenetic variations were recorded only in males, which become greener, less bright and saturated with growing size. Therefore, our results suggest the occurrence of two opposing strategies in colour expression between sexes: males' dorsal colouration plays a major role in communication, while females are more crypsis-oriented.

Close encounters of the three morphs: Does color affect aggression in a polymorphic lizard?

Color polymorphism is genetically controlled, and the process generating and maintaining morphs can affect speciation/extinction rates. Color badges are useful signals in intraspecific communication because they convey information about alternative strategies and can potentially decrease unnecessary conflicts among different color morphs. Competition and aggressive interactions among color morphs can contribute to polymorphism maintenance. This could lead to an uneven spatial distribution of morphs in a population because the local frequency of each morph establishes the intensity of the competition and the fitness of each male. We used a polymorphic lizard, Podarcis muralis, to assess if aggression varies among morphs under two contrasting hypotheses: a heteromorphic versus homomorphic aggression. We used laboratory mirror tests after lizard color manipulation, and we verified the consistency of results with an analysis of the spatial distribution of morphs in a wild population. Both the experiments confirmed that aggression is more common during homomorphic than heteromorphic contests. The adoption of alternative behavioral strategies that minimize risks and costs could facilitate the stable coexistence of the phenotypes and reduce competition. A bias in aggression would advantage rarer morph, which would suffer less harassment by common morphs obtaining a fitness advantage. This process would be negatively-frequency-dependent and would stabilize polymorphism, possibly contributing to sympatric speciation.

A simple lung ultrasound protocol for the screening of COVID-19 pneumonia in the emergency department.

The most relevant manifestation of coronavirus disease 2019 (COVID-19) is interstitial pneumonia. Several lung ultrasound (US) protocols for pneumonia diagnosis are used in clinical practice, but none has been proposed for COVID-19 patients' screening in the emergency department. We adopted a simplified 6-scan lung US protocol for COVID-19 pneumonia diagnosis (LUSCOP) and compared its sensitivity with high resolution computed tomography (HRCT) in patients suspected for COVID-19, presenting to one Emergency Department from February 21st to March 15th, 2020, during the outbreak burst in northern Italy. Patients were retrospectively enrolled if both LUSCOP protocol and HRCT were performed in the Emergency Department. The sensitivity of LUSCOP protocol and HRCT were compared. COVID-19 pneumonia's final diagnosis was based on real-time reverse-transcription polymerase chain reaction from nasal-pharyngeal swab and on clinical data. Out of 150 suspected COVID-19 patients, 131 were included in the study, and 130 had a final diagnosis of COVID-19 pneumonia. The most frequent lung ultrasonographic features were: bilateral B-pattern in 101 patients (77%), B-pattern with subpleural consolidations in 26 (19.8%) and lung consolidations in 2 (1.5%). LUSCOP Protocol was consistent with HRCT in correctly screening 130 out of the 131 COVID-19 pneumonia cases (99.2%). In one case COVID-19 pneumonia was excluded by both HRCT and lung US. LUSCOP protocol showed optimal sensitivity and can be proposed as a simple screening tool for COVID-19 pneumonia diagnosis in the context of outbreak burst areas where prompt isolation of suspected patients is crucial for patients' and operators' safety.

Morph-specific seasonal variation of aggressive behaviour in a polymorphic lizard species.

The persistence of colour polymorphism (CP) within a given population is generally associated with the coexistence of alternative reproductive strategies, each one involving specific trade-offs among behavioural, morphological, physiological, and other life histories. Common wall lizard (Podarcis muralis), is a medium-sized diurnal lizard, showing CP in three main colours (yellow, white, and red) on throat and belly, and a morph-specific pattern for both immunocompetence and seasonal variation of T levels. Yellow males show low stamina with high plasma T levels at the beginning of the season, while white males show high stamina with a higher plasma T levels at the end of the season. We hypothesised the presence of two strategies: a risky one, characterised by high aggressiveness played by yellow-morph, and a conservative one by white morph with low aggressiveness. Thus, we tested the aggressive response to conspecifics of yellow and white morphs using a mirror inserted into their cage, mimicking an intrusion of a stranger in their territories, throughout the breeding season (from April to July, 117 trials). We considered three types of aggressive response, with different levels of aggressiveness: (i) bite against the image reflected in the mirror, (ii) seconds spent by the individuals into the half mirrored cage, and (iii) number of times the lizard entered the half mirrored cage. We also considered the number of tongue flicking as explorative behaviour variable. All lizards were tested after a period of acclimatisation to the captivity conditions. Results demonstrate that yellow males showed a higher aggressive response in the early season and a decrease aggressive response towards the end, whereas white males showed an opposite pattern.

Statistical methodology for the evaluation of leukocyte data in wild reptile populations: A case study with the common wall lizard (Podarcis muralis).

The leukocyte profile has the potential to be a reliable method to measure health conditions and stress in wild animals, but limitations occur because current knowledge on reference intervals is largely incomplete, especially because data come from studies on captive animals involving few individuals from single populations. Here we propose a general framework for achieving reliable leukocyte reference intervals, encompassing a set of internal and external factors, potentially affecting the leukogram. To do so, we present a systematic survey of the hematology of the common wall lizard, Podarcis muralis, involving 794 lizards from 54 populations over the whole geographic range of the species in Italy. Reference intervals for white blood cell (WBC) and leukocyte differential count were obtained by using linear mixed models in a Bayesian framework. The application of the procedure clearly showed that both internal (sex and size) and external (latitude and season) factors are a source of variation of leukocyte profile. Furthermore, the leukogram of common wall lizard has a strong variability among populations, which accounts for more than 50% of the whole variation. Consequently, some common assumptions used in studies on captive individuals are no longer supported in wild populations, namely, i) any group of individuals is a representative sample, ii) any population is representative of all others, iii) geographic clines do not occur over the species range, and iv) seasonal variation has limited effects. We encourage researchers aimed at the definition of leukocyte reference intervals for wild populations of reptiles to involve a large number of populations over a wide geographic range in ad hoc statistical models to disentangle local and geographic effects on leukocyte profile variation.

Climate migrants' survival threatened by "C" shaped anthropic barriers.

Despite studies on range shifts being abundant, the problem of dispersal barriers limiting climate migrants' movement is yet to be fully included into any modeling framework. For this reason, we introduce a novel concept whereby the interplay of range shifts and dispersal barriers of a particular spatial configuration can threaten the persistence of populations under a climate change scenario. We named this concept "C-trap," based on the topographic shape of such barriers. After elaborating on the theoretical features of C-traps, we provide a simple method that combines environmental data and future climate projections to locate them spatially. We use this method to determine where high C-trap densities have the potential to further threaten the conservation of endangered, endemic animals across the world's terrestrial realm, in a climate change scenario. Our methodology detected potential C-traps for the study system, with areas of high density mostly located in east Europe, south Asia and North America. However, finer-scale analyses are required to assess the magnitude of the threat locally. Dispersal barriers add an additional dimension to range shift studies and can ultimately prevent otherwise successful climate migrants from tracking their climatic niche. The methodology presented here is simple and flexible enough to be adapted to a wide range of taxa and locations as well as the fast development of range shift modeling. Therefore, we encourage researchers to include the effects of anthropogenic dispersal barriers in range shifts models and in the planning of effective conservation strategies with reference to climate change.

Seasonal Variations in Femoral Gland Secretions Reveals some Unexpected Correlations Between Protein and Lipid Components in a Lacertid Lizard.

Animals modulate intraspecific signal shape and intensity, notably during reproductive periods. Signal variability typically follows a seasonal scheme, traceable through the expression of visual, acoustic, chemical and behavioral patterns. The chemical channel is particularly important in lizards, as demonstrated by well-developed epidermal glands in the cloacal region that secrete lipids and proteins recognized by conspecifics. In males, the seasonal pattern of gland activity is underpinned by variation of circulating androgens. Changes in the composition of lipid secretions convey information about the signaler's quality (e.g., size, immunity). Presumably, individual identity is associated with a protein signature present in the femoral secretions, but this has been poorly investigated. For the first time, we assessed the seasonal variability of the protein signal in relation to plasma testosterone level (T), glandular activity and the concentration of provitamin D3 in the lipid fraction. We sampled 174 male common wall lizards (Podarcis muralis) over the entire activity season. An elevation of T was observed one to two months before the secretion peak of lipids during the mating season; such expected delay between hormonal fluctuation and maximal physiological response fits well with the assumption that provitamin D3 indicates individual quality. One-dimensional electrophoretic analysis of proteins showed that gel bands were preserved over the season with an invariant region; a result in agreement with the hypothesis that proteins are stable identity signals. However, the relative intensity of bands varied markedly, synchronously with that of lipid secretion pattern. These variations of protein secretion suggest additional roles of proteins, an issue that requires further studies.

Morph-specific protein patterns in the femoral gland secretions of a colour polymorphic lizard.

Colour polymorphism occurs when two or more genetically-based colour morphs permanently coexist within an interbreeding population. Colouration is usually associated to other life-history traits (ecological, physiological, behavioural, reproductive …) of the bearer, thus being the phenotypic marker of such set of genetic features. This visual badge may be used to inform conspecifics and to drive those decision making processes which may contribute maintaining colour polymorphism under sexual selection context. The importance of such information suggests that other communication modalities should be recruited to ensure its transfer in case visual cues were insufficient. Here, for the first time, we investigated the potential role of proteins from femoral gland secretions in signalling colour morph in a polymorphic lizard. As proteins are thought to convey identity-related information, they represent the ideal cues to build up the chemical modality used to badge colour morphs. We found strong evidence for the occurrence of morph-specific protein profiles in the three main colour-morphs of the common wall lizard, which showed both qualitative and quantitative differences in protein expression. As lizards are able to detect proteins by tongue-flicking and vomeronasal organ, this result support the hypothesis that colour polymorphic lizards may use a multimodal signal to inform about colour-morph.

Towards mm-wave spectroscopy for dielectric characterization of breast surgical margins.

PURPOSE: The evaluation of the surgical margin in breast conservative surgery is a matter of general interest as such treatments are subject to the critical issue of margin status as positive surgical margins can undermine the effectiveness of the procedure. The relatively unexplored ability of millimeter-wave (mm-wave) spectroscopy to provide insight into the dielectric properties of breast tissues was investigated as a precursor to their possible use in assessment of surgical margins. METHODS: We assessed the ability of a mm-wave system with a roughly hemispherical sensitive volume of ∼3 mm radius to distinguish malignant breast lesions in prospectively and consecutively collected tumoral and non-tumoral ex-vivo breast tissue samples from 91 patients. We characterized the dielectric properties of 346 sites in these samples, encompassing malignant, fibrocystic disease and normal breast tissues. An expert pathologist subsequently evaluated all measurement sites. RESULTS: At multivariate analysis, mm-wave dielectric properties were significantly correlated to histologic diagnosis and fat content. Further, using 5-fold cross-validation in a Bayesian logistic mixed model that considered the patient as a random effect, the mm-wave dielectric properties of neoplastic tissues were significantly different from normal breast tissues, but not from fibrocystic tissue. CONCLUSION: Reliable discrimination of malignant from normal, fat-rich breast tissue to a depth compatible with surgical margin assessment requirements was achieved with mm-wave spectroscopy.

A new method for modelling biological invasions from early spread data accounting for anthropogenic dispersal.

Biological invasions are one of the major causes of biodiversity loss worldwide. In spite of human aided (anthropogenic) dispersal being the key element in the spread of invasive species, no framework published so far accounts for its peculiar characteristics, such as very rapid dispersal and independence from the existing species distribution. We present a new method for modelling biological invasions using historical spatio-temporal records. This method first discriminates between data points of anthropogenic origin and those originating from natural dispersal, then estimates the natural dispersal kernel. We use the expectation-maximisation algorithm for the first step; we then use Ripley's K-function as a spatial similarity metric to estimate the dispersal kernel. This is done accounting for habitat suitability and providing estimates of the inference precision. Tests on simulated data show good accuracy and precision for this method, even in the presence of challenging, but realistic, limitations of data in the invasion time series, such as gaps in the survey times and low number of records. We also provide a real case application of our method using the case of Litoria frogs in New Zealand. This method is widely applicable across the field of biological invasions, epidemics and climate change induced range shifts and provides a valuable contribution to the management of such issues. Functions to implement this methodology are made available as the R package Biolinv (https://cran.r-project.org/package=Biolinv).

Morph-specific assortative mating in common wall lizard females.

Color polymorphism often is associated with alternative reproductive strategies and may reflect different adaptive optima that coexist within populations. The equilibrium among morph frequencies is maintained by the occurrence of opposite selective pressures (disruptive vs. stabilizing), which promote polymorphism while preserving gene flow. Sexual selection may contribute on both sides, particularly when morphs do not mate randomly. Reptiles offer a good model, notably lizards. Nevertheless, previous studies on mate choice in polymorphic lizards have generated contrasting results, with some studies suggesting that female morphs might tune their preference depending on environmental/social conditions such as crowding. We experimentally manipulated the number of individuals a female common wall lizard Podarcis muralis perceives around her, to test if females of different morphs (white or yellow) tune their choice for white and yellow males in order to maximize the probability that hatchlings follow the strategy best adapted to the population density. Results showed that crowding experienced by females did not affect mate choice, arguing against a flexible choice strategy by females. However, white females significantly associated with white males, whereas yellow females did not significantly associate with yellow males. Thus, sexual selection could contribute to the maintenance of color polymorphism in this species by a mix of assortative and non-assortative mating strategies, which could maintain the equilibrium between gene divergence and gene flow among morphs.

Inter- and intra-population variability of the protein content of femoral gland secretions from a lacertid lizard.

Femoral glands of male lizards produce waxy secretions that are involved in inter- and intraspecific chemical communication. The main components of these secretions are proteins and lipids, the latter having been extensively studied and already associated to male quality. On the opposite, the composition and role of proteins are nearly unknown, the only available information coming from few studies on iguanids. These studies got the conclusion that proteins might have a communicative function, notably they could signal individual identity. A generalization of these findings requires the extension of protein analysis to other lizard families, and the primary detection of some patterns of individual variability. Using the common wall lizard Podarcis muralis as a model species, the protein fraction of the femoral pore secretions was investigated to provide the first characterization of this component in a lacertid lizard and to explore its source of variability, as a first step to support the hypothesized communicative role. Samples of proteins from femoral secretions were collected from 6 Italian populations and subjected to 1-dimensional electrophoresis. The binary vector of the band presence/absence was used to define the individual profiles. Protein fraction is found to have a structured pattern, with both an individual and a population component. Although the former supports the potential communicative role of proteins, the latter offers a double interpretation, phylogenetic or environmental, even though the phylogenetic effect seems more likely given the climatic resemblance of the considered sites. Further studies are necessary to shed light on both these issues.

Seasonal variations of plasma testosterone among colour-morph common wall lizards (Podarcis muralis).

Sexual steroids influence reproductive behaviours and promote secondary sexual traits. In male lizards, increasing levels of testosterone (T) bolster conspicuous colouration, stimulate territoriality, and trigger antagonistic interactions among rivals. Moreover, in colour polymorphic species, reproductive strategy, aggressiveness and T levels can differ between morphs. Therefore, T level is considered as an important mechanism that regulates the expression of colour polymorphism and sexual behaviours of males. But in the polymorphic territorial wall lizard (Podarcis muralis), a lack of relationship between morphs and aggressiveness challenges the notion that T plays such a role. To examine this issue, we compared adult T levels among three colour morphs (white, yellow and red) through repeated sampling during the mating season. High T levels were observed at the onset of the mating season followed by a significant decrease, a pattern documented in other lizard species. Mean T levels did not differ among morphs. However, yellow males maintained significantly higher T levels over time and displayed a stronger subsequent decline. Overall, in this species, seasonal T patterns differ among morphs, not mean values. Previous studies revealed that T suppresses the immune response; suggesting that a strong initial investment promoted by high T levels may trade-off against immunity (maintenance). Further experimental investigations are required to clarify the relationship between T and reproductive effort in polymorphic species that exhibit complex temporal pattern of T levels.

Assessing the Spatial Scale Effect of Anthropogenic Factors on Species Distribution.

Patch context is a way to describe the effect that the surroundings exert on a landscape patch. Despite anthropogenic context alteration may affect species distributions by reducing the accessibility to suitable patches, species distribution modelling have rarely accounted for its effects explicitly. We propose a general framework to statistically detect the occurrence and the extent of such a factor, by combining presence-only data, spatial distribution models and information-theoretic model selection procedures. After having established the spatial resolution of the analysis on the basis of the species characteristics, a measure of anthropogenic alteration that can be quantified at increasing distance from each patch has to be defined. Then the distribution of the species is modelled under competing hypotheses: H0, assumes that the distribution is uninfluenced by the anthropogenic variables; H1, assumes the effect of alteration at the species scale (resolution); and H2, H3 … Hn add the effect of context alteration at increasing radii. Models are compared using the Akaike Information Criterion to establish the best hypothesis, and consequently the occurrence (if any) and the spatial scale of the anthropogenic effect. As a study case we analysed the distribution data of two insular lizards (one endemic and one naturalised) using four alternative hypotheses: no alteration (H0), alteration at the species scale (H1), alteration at two context scales (H2 and H3). H2 and H3 performed better than H0 and H1, highlighting the importance of context alteration. H2 performed better than H3, setting the spatial scale of the context at 1 km. The two species respond differently to context alteration, the introduced lizard being more tolerant than the endemic one. The proposed approach supplies reliably and interpretable results, uses easily available data on species distribution, and allows the assessing of the spatial scale at which human disturbance produces the heaviest effects.