Plastic and genomic change of a newly established lizard population following a founder event.

Understanding how phenotypic divergence arises among natural populations remains one of the major goals in evolutionary biology. As part of competitive exclusion experiment conducted in 1971, 10 individuals of Italian wall lizard (Podarcis siculus (Rafinesque-Schmaltz, 1810)) were transplanted from Pod Kopiste Island to the nearby island of Pod Mrcaru (Adriatic Sea). Merely 35 years after the introduction, the newly established population on Pod Mrcaru Island had shifted their diet from predominantly insectivorous towards omnivorous and changed significantly in a range of morphological, behavioural, physiological and ecological characteristics. Here, we combine genomic and quantitative genetic approaches to determine the relative roles of genetic adaptation and phenotypic plasticity in driving this rapid phenotypic shift. Our results show genome-wide genetic differentiation between ancestral and transplanted population, with weak genetic erosion on Pod Mrcaru Island. Adaptive processes following the founder event are indicated by highly differentiated genomic loci associating with ecologically relevant phenotypic traits, and/or having a putatively adaptive role across multiple lizard populations. Diverged traits related to head size and shape or bite force showed moderate heritability in a crossing experiment, but between-population differences in these traits did not persist in a common garden environment. Our results confirm the existence of sufficient additive genetic variance for traits to evolve under selection while also demonstrating that phenotypic plasticity and/or genotype by environment interactions are the main drivers of population differentiation at this early evolutionary stage.

Relationships between dietary breadth and flexibility in jaw movement: A case study of two recently diverged insular populations of Podarcis lizards.

The kinematics of lizard feeding are the result of complex interactions between the craniocervical, the hyolingual, and the locomotor systems. The coordinated movement of these elements is driven by sensory feedback from the tongue and jaws during intraoral transport. The kinematics of jaw movements have been suggested to be correlated with the functional characteristics of the prey consumed, such as prey mobility and hardness. However, whether and how dietary breadth correlates with the flexibility in the behavioral response has rarely been tested, especially at the intraspecific level. Here we tested whether an increase in dietary breadth was associated with a greater behavioral flexibility by comparing two recently diverged populations of insular Podarcis lizards differing in dietary breadth. To do so, we used a stereoscopic high-speed camera set-up to analyze the jaw kinematics while offering them different prey types. Our results show that prey type impacts kinematics, especially maximum gape, and maximum opening and closing speed. Furthermore, the behavioral flexibility was greater in the population with the greater dietary breadth, suggesting that populations which naturally encounter and feed on more diverse prey items show a greater ability to modulate their movements to deal with variation in functionally relevant prey properties. Finally, the more generalist population showed more stereotyped movements suggesting a finer motor control.

From micro to macroevolution: drivers of shape variation in an island radiation of Podarcis lizards.

Phenotypictraits have been shown to evolve in response to variation in the environment. However, the evolutionary processes underlying the emergence of phenotypic diversity can typically only be understood at the population level. Consequently, how subtle phenotypic differences at the intraspecific level can give rise to larger-scale changes in performance and ecology remains poorly understood. We here tested for the covariation between ecology, bite force, jaw muscle architecture, and the three-dimensional shape of the cranium and mandible in 16 insular populations of the lizards Podarcis melisellensis and P. sicula. We then compared the patterns observed at the among-population level with those observed at the interspecific level. We found that three-dimensional head shape as well as jaw musculature evolve similarly under similar ecological circumstances. Depending on the type of food consumed or on the level of sexual competition, different muscle groups were more developed and appeared to underlie changes in cranium and mandible shape. Our findings show that the local selective regimes are primary drivers of phenotypic variation resulting in predictable patterns of form and function. Moreover, intraspecific patterns of variation were generally consistent with those at the interspecific level, suggesting that microevolutionary variation may translate into macroevolutionary patterns of ecomorphological diversity.

Differences in cautiousness between mainland and island Podarcis siculus populations are paralleled by differences in brain noradrenaline/adrenaline concentrations.

Adaptive behavior is shaped by the type and intensity of selection pressures coming from the environment, such as predation risk and resource availability, and can be modulated by individual's neuroendocrine profile involving steroid hormones and the brain-stem monoaminergic circuits projecting to forebrain structures. Boldness when faced with a predator and exploration/activity when confronted with a new environment reflect the degree of cautiousness and/or "risk-taking" of an individual. In this study we have explored to which extent two populations of Podarcis siculus occupying different ecological niches: mainland (ML) and an islet (ISL) differ in the level of cautiousness and whether these differences are paralleled by differences in their monoaminergic profiles. Boldness was tested in the field as antipredator behavior, while novel space and object explorations were tested in a laboratory setting in an open field apparatus. Finally, serotonin, dopamine, noradrenaline (NA) and adrenaline (ADR) concentrations were measured in whole brain samples by ELISA. Lizards from ML population spent significantly more time hiding after a predator encounter in the field, displayed lower intensity of novel space exploration in a laboratory setting, and contained significantly higher whole-brain concentrations of NA and ADR than their ISL counterparts. Parallelism between the level of risk-taking behavior and concentrations of neurotransmitters mediating alertness and reaction to stress suggests that the differing environmental factors on ML and ISL may have shaped the degree of cautiousness in the residing lizard populations by affecting the activity of NA/ADR neural circuits.

Morphological and ecological divergence in two populations of European glass lizard, Pseudopus apodus (Squamata: Anguidae).

The European glass lizard, Pseudopus apodus (Pallas, 1775), is a large, legless lizard with wide distribution across south-eastern Europe and eastern and central Asia. To date, morphological diversification among populations on a geographically small scale has not yet been reported in this lizard. Thus, we investigated the morphological variations and corresponding differences in habitat utilization between two populations of P. apodus inhabiting the same biogeographical zone within a relatively close geographic area. We hypothesized that minor differences in habitat could induce a significant level of morphological differentiation, thus indicating morphological plasticity in this species on a small geographical scale. We sampled 164 individuals (92 from the Croatian mainland and 72 from the island of Cres). Results showed that P. apodus indeed exhibited morphological differences between populations in the same biogeographical zone within a relatively close geographic area, with the Cres Island individuals being generally larger than the individuals from the mainland. Some ecological characteristics were similar in both populations (e.g., soil temperature, distance to hiding place), whereas others were distinct (e.g., air temperature and humidity). In addition, vegetation cover differed between the two sites, with more vegetation present on the mainland than on the island. Furthermore, the Cres Island population showed clear sexual dimorphism, which was absent in the mainland population.

Diet variability among insular populations of Podarcis lizards reveals diverse strategies to face resource-limited environments.

Access to resources is a dynamic and multicausal process that determines the success and survival of a population. It is therefore often challenging to disentangle the factors affecting ecological traits like diet. Insular habitats provide a good opportunity to study how variation in diet originates, in particular in populations of mesopredators such as lizards. Indeed, high levels of population density associated with low food abundance and low predation are selection pressures typically observed on islands. In the present study, the diet of eighteen insular populations of two closely related species of lacertid lizards (Podarcis sicula and Podarcis melisellensis) was assessed. Our results reveal that despite dietary variability among populations, diet taxonomic diversity is not impacted by island area. In contrast, however, diet disparity metrics, based on the variability in the physical (hardness) and behavioral (evasiveness) properties of ingested food items, are correlated with island size. These findings suggest that an increase in intraspecific competition for access to resources may induce shifts in functional components of the diet. Additionally, the two species differed in the relation between diet disparity and island area suggesting that different strategies exist to deal with low food abundance in these two species. Finally, sexual dimorphism in diet and head dimensions is not greater on smaller islands, in contrast to our predictions.

Coexisting lacertid lizard species Podarcis siculus and Podarcis melisellensis differ in dopamine brain concentrations.

In the eastern Adriatic, Podarcis siculus, an invasive species, competitively excludes the native Podarcis melisellensis. Monoamine neurotransmitters-serotonin (5HT), dopamine (DA), and noradrenaline (NA)-are implicated in social behavior, and could lie at the basis of the direct behavioral interference of P. siculus with P. melisellensis. To understand the relationship between social behavior and monoamines, as well as the differences in behavior between P. siculus and P. melisellensis, we developed a high-performance liquid chromatography (UV/VIS detection) method with which we were able to reliably measure concentrations of 5HT, DA, and NA in 32 brains of the two lizard species. We observed no statistically significant influence of species, sex, or their interaction on brain NA and 5HT concentrations. Statistically significant influence of species on dopamine levels were recorded, with P. siculus having twice as much dopamine in their brains. Taking into account that a significant aggressive relationship, with P. siculus dominating over P. melisellensis, has been previously observed, and that dopamine directly influences this behavior, the observed differences in dopamine levels could represent a trait in these species and may contribute to the competitive exclusion of P. melisellensis by P. siculus in the eastern Adriatic.

Population-specific effects of developmental temperature on body condition and jumping performance of a widespread European frog.

All physiological processes of ectotherms depend on environmental temperature. Thus, adaptation of physiological mechanisms to the thermal environments is important for achieving optimal performance and fitness. The European Common Frog, Rana temporaria, is widely distributed across different thermal habitats. This makes it an exceptional model for studying the adaptations to different thermal conditions. We raised tadpoles from Germany and Croatia at two constant temperature treatments (15°C, 20°C), and under natural temperature fluctuations (in outdoor treatments), and tested how different developmental temperatures affected developmental traits, that is, length of larval development, morphometrics, and body condition, as well as jumping performance of metamorphs. Our results revealed population-specific differences in developmental time, body condition, and jumping performance. Croatian frogs developed faster in all treatments, were heavier, in better body condition, and had longer hind limbs and better jumping abilities than German metamorphs. The populations further differed in thermal sensitivity of jumping performance. While metamorphs from Croatia increased their jumping performance with higher temperatures, German metamorphs reached their performance maximum at lower temperatures. These population-specific differences in common environments indicate local genetic adaptation, with southern populations being better adapted to higher temperatures than those from north of the Alps.

Addition of propolis to irinotecan therapy prolongs survival in ehrlich ascites tumor-bearing mice.

We investigated possible synergistic action of anticancer drug Irinotecan (IRI) combined with ethanolic (EEP) and water-soluble (WSDP) derivate of propolis on Swiss albino mice injected with Ehrlich ascites tumor (EAT). For survival analysis mice were administered WSDP and EEP (100 mg/kg) daily for 3 consecutive days, beginning on 3rd day after EAT cell (1×106) injection. IRI was administered at a dose of 50 mg/kg on days 1, 13, and 19. We simultaneously studied peripheral white blood cell count, cell types washed from the peritoneal cavity, functional activity of macrophages from peritoneal cavity, and the level of primary DNA damage in leukocytes, kidney, and liver cells using the alkaline comet assay. Three out of 9 mice per group survived the entire duration of the experiment (90 days) in groups treated with IRI combined with WSDP and EEP. All test components increased survival of mice by 7.53% to 231.54%. Combined treatment with IRI and/or WSDP and EEP significantly decreased percentage of tumor cells in the peritoneal cavity as compared to nontreated EAT-injected mice. All treated animals had significantly higher percentage of neutrophils in the peritoneal cavity in comparison to nontreated EAT-injected mice. We observed significantly higher value of DNA damage in leukocytes of mice treated with IRI and combination of IRI and/or WSDP and EEP as compared to nontreated EAT-injected mice, while the same treatment decreased DNA damage in kidney. Our results showed that addition of propolis to IRI treatment enhanced antitumor activity of IRI and prolongs survival in EAT-bearing mice, which definitely deserve further studies to clarify the possible mechanisms of antitumor actions of combined herb-drug treatments.

Role of flavonoids on oxidative stress and mineral contents in the retinoic acid-induced bone loss model of rat.

PURPOSE: Reactive oxygen species play a role in a number of degenerative conditions including osteoporosis. Flavonoids as phyto-oestrogens exert physiological effects against oxidative stress diseases. We developed a retinoic acid-induced bone loss model of rats to assess whether flavonoids and alendronate as positive control have role against oxidative stress and mineral contents in osteoporosis in vivo. METHODS: Three-month-old female rats of the Y59 strain were given quercetin, chrysin, naringenin (100 mg kg(-1)) or alendronate (40 mg kg(-1), a positive control) immediately before retinoic acid treatment (80 mg kg(-1)) once daily for 14 days by a single intragastric (i.g.) application. In the second part of the study, we assessed the effect of those flavonoids on the skeletal system of healthy rats using single i.g. application on the respective flavonoids during 14 days. Twenty-four hours after the treatment, we analysed bone mineral density and the total content of bone calcium and phosphorus in the femur, the geometric and physical characteristics of thigh bones and lipid peroxidation and glutathione levels of liver and kidney cells. RESULTS: All flavonoids improved the decrease in bone weight coefficient, the length and the diameter of the bone, the content of bone ash and calcium and phosphorus content induced by retinoic acid. Chrysin and quercetin showed promise as preventive agents. Flavonoids were superior to alendronate according to some criteria. CONCLUSIONS: These results suggest that the dietary flavonoids could reduce retinoic acid-induced oxidative stress and bone loss and that flavonoids may be useful therapeutics for prevention of skeletal diseases.

Synergism between propolis and hyperthermal intraperitoneal chemotherapy with cisplatin on ehrlich ascites tumor in mice.

We investigated antitumor, genotoxic, chemopreventive, and immunostimulative effects of local chemoimmunotherapy and hyperthermal intraperitoneal chemotherapy (HIPEC) in a mouse-bearing Ehrlich ascites tumor (EAT). Mice were treated with water-soluble derivative of propolis (WSDP) at a dose of 50 mg kg(-1) , 7 and 3 days before implantation of EAT cells, whereas cisplatin (5 or 10 mg kg(-1) ) was injected 3 days after implantation of EAT cells at 37°C and 43°C. The following variables were analyzed: the total number of cells, differential count of the cells present in the peritoneal cavity, functional activity of macrophages, comet assay, and micronucleus assay. The combination of WSDP + CIS 5 mg kg(-1) at 37°C resulted in tumor growth inhibition and increased the survival of mice by additional 115.25%. WSDP with HIPEC increased the survival of mice by additional 160.3% as compared with HIPEC. WSDP reduced cisplatin toxic and genotoxic effect to normal cells without affecting cisplatin cytotoxicity on EAT cells. In addition, WSDP with HIPEC increased the cytotoxic actions of macrophages to tumor cells. Water-soluble derivative of propolis increases macrophage activity and sensitivity of tumor cells to HIPEC and reduces cisplatin toxicity to normal cells.

Synergistic effects of irinotecan and flavonoids on Ehrlich ascites tumour-bearing mice.

Swiss albino mice were given Ehrlich ascites tumour cells (1 × 10(6)) intraperitoneally. For survival analysis and tumour growth analysis, the mice were administered quercetin and naringin (100 mg/kg) daily for 3 consecutive days, beginning on the third day after intraperitoneal (i.p.) injection of Ehrlich ascites tumour cells (1 × 10(6)). Irinotecan was administered ip at a dose of 50 mg/kg on days 1, 13 and 19. For the analysis of cell types and differential count of cells present in the peritoneal cavity, peripheral whole-blood leucocyte count and the comet assay, the mice were treated therapeutically with quercetin and naringin (100 mg/kg) and irinotecan (50 mg/kg) daily for 3 consecutive days beginning on third day after i.p. injection of Ehrlich ascites tumour cells (1 × 10(6)). We observed the synergistic anti-tumour effect expressed as the median survival time of mice treated with naringin in combination with irinotecan. All test components inhibited tumour growth and increased lifespan of mice except quercetin. The total number of cells present in the peritoneal cavity of mice significantly decreased in all treatments except quercetin. Single irinotecan and irinotecan combined with naringin had the highest DNA-damaging potential on peripheral blood leucocytes and lowest primary DNA damage, both in the kidney and liver cells as measured by the alkaline comet assay. Our results showed enhanced anti-tumour activity of irinotecan in combined treatment with flavonoids to reduce the deteriorating reaction of cytostatic drugs.

Fast and furious: effects of body size on strike performance in an arboreal viper Trimeresurus (Cryptelytrops) albolabris.

Body size has a pervasive effect on animal functioning and life history with size dependent changes in performance and physiology throughout ontogeny being common in many ectothermic vertebrates. However, as selection on juvenile life history stages is strong, juveniles often offset the disadvantages of small body size by disproportionate levels of performance. Here, we investigate size-related changes in defensive strike performance in an arboreal pit viper, Trimerusurus (Cryptelytrops) albolabris. Our data show a significant negative allometry in the scaling of head dimensions and head mass to body mass. However, strike velocity and strike distance are independent of body mass, with juveniles in our sample striking as fast and as far as adults. In contrast to model predictions suggesting that acceleration capacity should decrease with increasing body mass, acceleration capacity increases with snake body mass. Our results suggest that this is the result of a negative allometric scaling of head mass combined with an isometric scaling of the dorsal epaxial musculature. Finally, our data show a significant sexual dimorphism in body size and strike velocity with females being heavier and striking faster independent of the dimorphism in body size.

Evaluation of micronucleus and erythrocytic nuclear abnormalities in Balkan whip snake Hierophis gemonensis.

Over recent years, changes of erythrocytic nuclei have been increasingly used to evaluate genotoxic effects of different compounds such as polycyclic aromatic hydrocarbons (benzo-a-pyrene, naphthalene, ß-naphthoflavone), heavy metals (cadmium, mercury), textile mill effluent especially in aquatic ecosystem. However, in fish, both micronuclei and erythrocytic nuclear abnormalities also appear spontaneously and their frequency can be seasonally dependent. The aim of the study was to evaluate the frequency of micronuclei (MN), nuclear abnormalities (NA) including vacuolated nuclei (VN) and cytoplasmic vacuoles (CV) in erythrocytes of Balkan whip snake Hierophis gemonensis and establish the level of spontaneous appearance during the annual cycle. Average frequency of NA was 10.89 ± 4.72% while the MN (0.03 ± 0.03%) and VN (0.04 ± 0.08%) were seldom detected. NA significantly positively correlated with MN (r = 0.319; P < 0.05) and VN (r = 0.363; P < 0.05). Appearance of CV did not correlate with other measured parameters and average frequency was 11.06 ± 8.33%. Significant seasonal variation was found in NA appearance with the lowest value in spring and the highest in winter. VN increase was observed in autumn. MN and CV levels varied between seasons but not significantly. Considering the biological cycle, frequency of NA, VN, MN and CV recorded in pre-hibernation/hibernation increased compared to the active phase, but only NA elevation was significant. Although the obtained results showed differences according to sex, statistical analysis of measured parameters showed the same pattern of seasonal variation in both sexes.

Brain toxicokinetics of prometryne in mice.

Prometryne is a methylthio-s-triazine herbicide. Significant trace amounts are found in the environment, mainly in water, soil, and food plants. The aim of this study was to establish brain and blood prometryne levels after single oral dose (1 g kg-1) in adult male and female mice. Prometryne was measured using the GC/MS assay at 1, 2, 4, 8, and 24 h after prometryne administration. Peak brain and blood prometryne values were observed 1 h after administration and they decreased in a time-dependent manner. Male mice had consistently higher brain and blood prometryne levels than female mice. The observed prometryne kinetics was similar to that reported for the structurally related herbicide atrazine.

Protective effects of propolis and related polyphenolic/flavonoid compounds against toxicity induced by irinotecan.

Despite the excellent chemotherapeutic effect of irinotecan, its cytotoxicity and genotoxicity in normal cells remains a major problem in chemotherapy. This study was carried out to find whether propolis preparations and related flavonoids (quercetin, naringin) might enhance irinotecan-induced cytotoxicity to tumor cells in mice bearing Ehrlich ascites tumors (EAT) while protecting normal blood, liver, and kidney cells. The preparation of propolis and their flavonoids were given to mice intraperitoneally at a dose of 100 mg kg(-1) body weight for three consecutive days before the ip injection of EAT cells (2×10(6)). Irinotecan was administered ip at dose of 50 mg kg(-1) on days 3, 4, and 5 after tumor cell inoculation. The combination treatment resulted in substantial inhibition of the growth of EAT cells as well as treatment with quercetin or irinotecan alone, whereas other treatment by itself showed little effect. However, when mice were pre-treated with test components prior to irinotecan, the frequencies of irinotecan-induced micronuclei (MN) was decreased but in mice bearing tumor QU and EEP increased number of micronucleated cells. Propolis preparation and related flavonoids were found to exhibit an important immunomodulatory effect and could decrease irinotecan-induced toxic and genotoxic effects to normal cells without effecting irinotecan cytotoxicity in EAT cells.

Radioprotective effects of quercetin and ethanolic extract of propolis in gamma-irradiated mice.

The aim of this study was to assess radioprotective effects of quercetin and the ethanolic extract of propolis (EEP) in CBA mice exposed to a single radiation dose 4 Gy (60Co). The mice were treated with 100 mg kg(-1) quercetin or EEP a day for three consecutive days either before (pre-treatment) or after gamma-irradiation (therapy). Leukocyte count was determined in blood drawn from the tail vein, and DNA damage in leukocytes was assessed using the alkaline comet assay. Genotoxic effects of the test compounds were also evaluated in non-irradiated mice. The levels of radioprotection provided by both test compounds were compared with those established in mice that were given chemical radioprotector S-(2-aminoethy1)isothiouronium bromide hydrobromide (AET). Mice that received pre-treatment were less sensitive to irradiation. Mice given the post-irradiation therapy showed a slight but not significant increase in total leukocyte count over irradiated negative control. Quercetin showed better protective properties than EEP in both pre-treatment and therapy, and activated a higher number of leukocytes in non-irradiated mice. The alkaline comet assay suggests that both natural compounds, especially when given as pre-treatment, protect against primary leukocyte DNA damage in mice. At tested concentrations, EEP and quercetin were not genotoxic to non-irradiated mice. AET, however, caused a slight but not significant increase in DNA damage. Although the results of this study show the radioprotective potential of the test compounds, further investigation is needed to clarify the underlying protection mechanisms.

Prevention of peritoneal carcinomatosis in mice with combination hyperthermal intraperitoneal chemotherapy and IL-2.

PURPOSE: The purpose of this study was to investigate the effect of local chemoimmunotherapy and hyperthermal intraperitoneal chemotherapy (HIPEC) in a mouse model of induced peritoneal carcinomatosis. MATERIAL AND METHODS: Peritoneal carcinomatosis in mice was produced by intraperitoneal implantation of MCa cells (5 x 10(3)). Interleukin-2 (4.1 x 10(4) IU/mouse) was injected into the abdominal cavity of mice at day 7 and 3 before implantation of tumour cells. Immediately after implantation of MCa cells mice were treated twice with 2 ml of saline that was heated either at 37 degrees C or 43 degrees C and cytostatics (doxorubicin 20 mg kg(-1), cisplatin 10 mg kg(-1), mitomycin 5 mg kg(-1), or 5-FU 150 mg kg(-1)). We followed the survival of animals and side effects appearing with different forms of treatment. RESULTS: Combined treatment with Interleukin-2 (IL-2) and cytostatics (5-FU, CIS or MIT) significantly affected the development of peritoneal carcinomatosis and increased the survival of mice (ILS% - 37 degrees C = 29.88, 199.32, and 108.52, ILS% - 43 degrees C = 62.69, 260.50, and 178.05, respectively). However, intraperitoneal chemotherapy on survival time of mice with DOX + IL-2 was ineffective as compared with DOX alone. CONCLUSION: We would like to stress that treatment with IL-2 prior to tumour diagnosis is not clinically practical, rather, the manuscript attempts to describe an experimental proof of principle. Results suggest the synergistic effect of hyperthermia, chemotherapy and immunotherapy; IL-2 significantly increases antitumor activity of hyperthermic chemotherapy and survival rate of mice with peritoneal carcinomatosis.