Convergent evolution of a blood-red nectar pigment in vertebrate-pollinated flowers.

Nearly 90% of flowering plants depend on animals for reproduction. One of the main rewards plants offer to pollinators for visitation is nectar. Nesocodon mauritianus (Campanulaceae) produces a blood-red nectar that has been proposed to serve as a visual attractant for pollinator visitation. Here, we show that the nectar's red color is derived from a previously undescribed alkaloid termed nesocodin. The first nectar produced is acidic and pale yellow in color, but slowly becomes alkaline before taking on its characteristic red color. Three enzymes secreted into the nectar are either necessary or sufficient for pigment production, including a carbonic anhydrase that increases nectar pH, an aryl-alcohol oxidase that produces a pigment precursor, and a ferritin-like catalase that protects the pigment from degradation by hydrogen peroxide. Our findings demonstrate how these three enzymatic activities allow for the condensation of sinapaldehyde and proline to form a pigment with a stable imine bond. We subsequently verified that synthetic nesocodin is indeed attractive to Phelsuma geckos, the most likely pollinators of Nesocodon We also identify nesocodin in the red nectar of the distantly related and hummingbird-visited Jaltomata herrerae and provide molecular evidence for convergent evolution of this trait. This work cumulatively identifies a convergently evolved trait in two vertebrate-pollinated species, suggesting that the red pigment is selectively favored and that only a limited number of compounds are likely to underlie this type of adaptation.

Digestibility of black soldier fly larvae (Hermetia illucens) fed to leopard geckos (Eublepharis macularius).

Black soldier fly (BSF) larvae have been marketed as an excellent choice for providing calcium to reptiles without the need of dusting or gut loading. However, previous studies have indicated that they have limited calcium digestibility and are deficient in fat soluble vitamins (A, D3, and E). In this feeding and digestibility trial, 24 adult male leopard geckos were fed one of three diets for 4 months: 1) whole, vitamin A gut loaded larvae; 2) needle pierced, vitamin A gut loaded larvae; or 3) whole, non-gut loaded larvae. Fecal output from the geckos was collected daily and apparent digestibility was calculated for dry matter, protein, fat, and minerals. There were no differences in digestibility coefficients among groups. Most nutrients were well digested by the leopard geckos when compared to previous studies, with the exception of calcium (digestibility co-efficient 43%), as the calcium-rich exoskeleton usually remained intact after passage through the GI tract. Biochemistry profiles revealed possible deficits occurring over time for calcium, sodium, and total protein. In regards to vitamin A digestibility, plasma and liver vitamin A concentrations were significantly higher in the supplemented groups (plasma- gut loaded groups: 33.38 ± 7.11 ng/ml, control group: 25.8 ± 6.72 ng/ml, t = 1.906, p = 0.04; liver- gut loaded groups: 28.67 ± 18.90 µg/g, control group: 14.13 ± 7.41 µg/g, t = 1.951, p = 0.03). While leopard geckos are able to digest most of the nutrients provided by BSF larvae, including those that have been gut loaded, more research needs to be performed to assess whether or not they provide adequate calcium in their non-supplemented form.

Evaluation of ß-carotene assimilation in leopard geckos (Eublepharis macularius).

Although leopard geckos (Eublepharis macularius) are commonly kept under human care, their vitamin requirements are largely unknown. Many invertebrate preys display a low vitamin A concentration; thus, gut-loading insects with vitamin A or carotenoids is a common practice. The objective of this prospective experimental study was to investigate whether dietary supplementation with ß-carotene, including prey gut-loading, leads to sufficient vitamin A hepatic storage and prevents epithelial squamous metaplasia development in leopard geckos. Ten clinically healthy female leopard geckos were randomly divided in two groups with various supplementations: a group receiving vitamin A supplementation and a group receiving ß-carotene. Insects were gut-loaded continuously with a supplement containing vitamin A or ß-carotene, depending on the group. Oral supplementation with cod liver oil or carrot juice was administered weekly to each lizard from "vitamin A group" and "carotenoid group" respectively. After 10 weeks of supplementation, surgical hepatic biopsies were obtained in three geckos of each group while the two remaining geckos were euthanized to undergo complete necropsy. Hepatic vitamin A concentration was determined for each lizard (n = 10) by ultra-performance liquid chromatography. Histopathology revealed hepatocellular vacuolization and vitellogenic follicles in five females. Epithelial squamous metaplasia was not observed in any of the geckos. Hepatic vitamin A concentration was significantly higher in the carotenoid-supplemented group than in the vitamin A-supplemented group (p = 0.03). Our results suggest that in leopard geckos, dietary supplementation with ß-carotene allows sufficient vitamin A hepatic storage.

Spontaneous otoacoustic emissions in teiid lizards.

SOAE from the last major lizard family not yet systematically investigated, the teiids, were collected from the genera Callopistes, Tupinambis and Cnemidophorus. Although their papillae show characteristics of the family Teiidae, the papillae differ both in their size and in the arrangement of uni- and bi-directional hair-cell areas. Among these three genera, Callopistes showed few (2 or 3) SOAE peaks, whereas the other two genera showed more (up to 6 per ear). In the absence of knowledge of the tonotopic maps, however, it was not possible to clearly relate the spectral patterns to the differences in papillar anatomy, suggesting that the determinants of these patterns may be more subtle than anticipated.

Maternal allocation of carotenoids to eggs in an Anolis lizard.

The maternal allocation of carotenoids to eggs has been widely documented and manipulated. However, it is often assumed that the sole adaptive value of this allocation is to increase offspring fitness. Because carotenoids can be pro-oxidants or antioxidants depending on their concentrations and their chemical environment (i.e. presence of other antioxidants), dams may need to dispose of excess carotenoids upon depletion of other antioxidants to prevent oxidative damage. Additionally, the amount of carotenoids deposited in eggs may be dependent on male traits such as quality and coloration. We evaluated these two non-mutually exclusive hypotheses for carotenoid allocation to eggs and assessed paternal effects by supplementing male and female brown anole lizards, Anolis sagrei, with dietary carotenoids or with a combination of carotenoids and vitamin C. We found significant differences in the antioxidant capacities of fertilized and unfertilized eggs produced by female lizards, but the treatment did not affect the antioxidant capacity or carotenoid content of eggs. However, the carotenoid concentration of unfertilized eggs from carotenoid-supplemented females was significantly higher than eggs from the control group. Male coloration and body size did not affect the antioxidant capacity or carotenoid content of the eggs. Carotenoids may be allocated to unfertilized eggs to offset oxidative damage to the dam, with a neutral effect on offspring, rather than to solely provide antioxidant benefits to offspring as has been widely assumed.

Oral and dermal diflubenzuron exposure causes a hypothalamic-pituitary-thyroid (HPT) axis disturbance in the Mongolian racerunner (Eremias argus).

Diflubenzuron (DFB) is a potential endocrine-disrupting chemical. However, its thyroid endocrine effect on reptiles has not been reported. In this study, immature lizards (Eremias argus) were exposed to 20 mg kg-1 DFB once a week for 42 days through oral or dermal routes. Their body weight, plasma thyroid hormone levels, thyroid gland histology and the transcription of hypothalamic-pituitary-thyroid (HPT) axis-related genes in different tissues were assessed to explore the effects of DFB on the HPT axis of lizards. The body weight decreased significantly only after the dermal exposure to DFB. Triiodothyronine (T3) to thyroxine (T4) ratio in the male plasma also significantly increased after the dermal exposure. After oral exposure, the activity of thyroid gland was positively related to the thyroid hormone levels. Furthermore, the alterations in thyroid hormone levels affected the HPT axis-related gene expression, which was tissue dependent and sexually selected. The thyroid hormone receptor genes (trα and trß) in the brain and thyroid were more sensitive to oral exposure. However, only the dermal treatment affected the trα, trß and type 2 deiodinase (dio2) genes in the male liver. These results suggest that DFB exposure caused sex-specific changes in the thyroid function of lizards, and the dermal treatment may be an important route for the risk assessment of reptiles.

Dietary constraints can preclude the expression of an honest chemical sexual signal.

Identifying the factors that underlie signal divergences remains challenging in studies of animal communication. Regarding the chemical signalling, different compounds can be found in some species but be absent in others. We hypothesized that if the costs that are associated with the expression of some compounds are too high, their presence in the signal may be restricted. However, these compounds may be expressed and be functional when those costs are relaxed. Vitamin E (α-tocopherol), a dietary compound with metabolic relevancy, acts as an honest chemical sexual signal in many lizards but no in others such as the Carpetan Rock lizard (Iberolacerta cyreni). We investigated whether dietary supplementation favours the expression of this vitamin in scents of I. cyreni. We show that dietary constraints can preclude the expression of vitamin E in chemical secretions of wild males because was expressed when it was experimentally provided in the diet. Vitamin E supplementation also heightened the immune response of males and increased the interest of their scent for females, highlighting the vitamin E as a chemical sexual signal in this species. We suggest that diet could decisively act as a driver of intra- and interspecific divergences in the chemical signalling of lizards.

Direct and indirect effects of petroleum production activities on the western fence lizard (Sceloporus occidentalis) as a surrogate for the dunes sagebrush lizard (Sceloporus arenicolus).

The dunes sagebrush lizard (Sceloporus arenicolus) is a habitat specialist of conservation concern limited to shin oak sand dune systems of New Mexico and Texas (USA). Because much of the dunes sagebrush lizard's habitat occurs in areas of high oil and gas production, there may be direct and indirect effects of these activities. The congeneric Western fence lizard (Sceloporus occidentalis) was used as a surrogate species to determine direct effects of 2 contaminants associated with oil and gas drilling activities in the Permian Basin (NM and TX, USA): herbicide formulations (Krovar and Quest) and hydrogen sulfide gas (H2S). Lizards were exposed to 2 concentrations of H2 S (30 ppm or 90 ppm) and herbicide formulations (1× or 2× label application rate) representing high-end exposure scenarios. Sublethal behavioral endpoints were evaluated, including sprint speed and time to prey detection and capture. Neither H2S nor herbicide formulations caused significant behavioral effects compared to controls. To understand potential indirect effects of oil and gas drilling on the prey base, terrestrial invertebrate biomass and order diversity were quantified at impacted sites to compare with nonimpacted sites. A significant decrease in biomass was found at impacted sites, but no significant effects on diversity. The results suggest little risk from direct toxic effects, but the potential for indirect effects should be further explored.

Sexually dimorphic expression of CREB binding protein in the green anole brain.

Green anoles are seasonally breeding lizards in which male sexual behavior is primarily regulated by an annual increase in testosterone. This hormone activates stereotyped behaviors, as well as morphological and biochemical changes in the brain, with greater effect in the breeding season than in the non-breeding season. This study is the first description of CREB binding protein (CBP) in the reptilian brain, and investigates the possibility that changes in CBP, an androgen receptor coactivator, may facilitate differences in responsiveness to testosterone across seasons. A portion of this gene was cloned for the green anole, and in situ hybridization was performed to examine the expression of CBP in the brains of gonadally intact male and female green anoles across breeding states. Additionally, hormonal regulation of CBP was evaluated across sex and season in animals that were gonadectomized and treated with testosterone or a control. Similar to other vertebrates, CBP was expressed at relatively high levels in steroid-sensitive brain regions. In the anole ventromedial amygdala, CBP mRNA levels were nearly twice as high in gonadally intact females compared to males. In contrast, CBP expression did not differ across seasons or hormone manipulation in this brain region. No significant effects were detected in the preoptic area or ventromedial hypothalamus. This pattern suggests that CBP might influence female-biased functions controlled by the ventromedial amygdala, but is not consistent with a role in mediating seasonal differences in responsiveness to testosterone in these areas associated with reproductive function.

The diet of free-roaming Australian Central Bearded Dragons (Pogona vitticeps).

The central bearded dragon (Pogona vitticeps) is one of the most popular pet lizards. However, little is known regarding their nutrient requirement, or their natural diet. Therefore, the stomach contents of 14 free-roaming P. vitticeps were determined by flushing. These stomach contents were described taxonomically, and analyzed for crude protein content as well as fatty acid content and composition. Most of the dry matter intake was in the form of animal material (61%) stemming from nine arthropod orders. The most abundant were alates of the termite Drepanotermes sp., accounting for 95% of the total number of prey items and more than half of the total dry matter (DM) intake. Plant material contributed 16% of the total DM intake. The diets were high in crude protein (41-50% DM) and the total fatty acid content was 14-27% of the DM intake. The main fatty acid was C18:1n9c (51-56% of total fatty acids), and polyunsaturated fatty acids (n3 and n6) comprised 6-8% of the total fat intake. Our data suggest that P. vitticeps is an opportunistic predator, which exploits the seasonal availability of prey. Based on our data and other studies, a diet consisting of several insect species, supplemented with leafy vegetables, rich in n3 FA's, would best resemble the expected natural diet of P. vitticeps.

What are carotenoids signaling? Immunostimulatory effects of dietary vitamin E, but not of carotenoids, in Iberian green lizards.

In spite that carotenoid-based sexual ornaments are one of the most popular research topics in sexual selection of animals, the antioxidant and immunostimulatory role of carotenoids, presumably signaled by these colorful ornaments, is still controversial. It has been suggested that the function of carotenoids might not be as an antioxidant per se, but that colorful carotenoids may indirectly reflect the levels of nonpigmentary antioxidants, such as melatonin or vitamin E. We experimentally fed male Iberian green lizards (Lacerta schreiberi) additional carotenoids or vitamin E alone, or a combination of carotenoids and vitamin E dissolved in soybean oil, whereas a control group only received soybean oil. We examined the effects of the dietary supplementations on phytohaemagglutinin (PHA)-induced skin-swelling immune response and body condition. Lizards that were supplemented with vitamin E alone or a combination of vitamin E and carotenoids had greater immune responses than control lizards, but animals supplemented with carotenoids alone had lower immune responses than lizards supplemented with vitamin E and did not differ from control lizards. These results support the hypothesis that carotenoids in green lizards are not effective as immunostimulants, but that they may be visually signaling the immunostimulatory effects of non-pigmentary vitamin E. In contrast, lizards supplemented with carotenoids alone have higher body condition gains than lizards in the other experimental groups, suggesting that carotenoids may be still important to improve condition.

Nonylphenol effects on the HPA axis of the bioindicator vertebrate, Podarcis sicula lizard.

Nonylphenol (NP) is an endocrine disruptor widely distributed in the environment. It accumulates in the lipids of living organisms and enters the human food chain. The main source of human exposure is expected to be food, drinking water and foodstuff contaminated through leaching from packaging or pesticide formulation applications. NP acts as an estrogenic compound and it is able to mimic the action of estradiol 17ß (E2) by binding to the estrogen receptor (ER). The aim of the present study was to investigate the NP effects on the hypothalamic-pituitary-adrenal gland (HPA) axis of the bioindicator Podarcis sicula lizard. A time-dependent stimulation of the HPA axis and variations of both catecholamine plasma levels were showed. Moreover, NP effects on adrenal gland morphology were evaluated by light and transmission electron microscopy. Clear morphological signs of adrenal gland stimulation such as an increase of steroidogenic cord diameter and vascularization, a strong escalation of adrenaline cell number and a decrease of noradrenaline cells were observed. The notably elevated levels of adrenal hormones suggested a permanent turning on of hypothalamic corticotropin releasing factor (CRF) secretion together with a lack of the negative feedback of HPA axis, perturbing systemic responses of the organism. Our data may help to predict the biological alterations induced by NP and to extend its impact upon adrenal function.

The role of heritable and dietary factors in the sexual signal of a Hispaniolan Anolis lizard, Anolis distichus.

The diversity of sexual signals is astounding, and divergence in these traits is believed to be associated with the early stages of speciation. An increasing number of studies also suggest a role for natural selection in driving signal divergence for effective transmission in heterogeneous environments. Both speciation and adaptive divergence, however, are contingent on the sexual signal being heritable, yet this often remains assumed and untested. It is particularly critical that the heritability of carotenoid-based sexual signals is investigated because such traits may instead be phenotypically plastic indicators of an individual's quality that exhibit no or little heritable variation. We present the first study to investigate the relative contribution of genetic and environmental factors to the striking diversity of dewlap color and pattern in Anolis lizards. Using a breeding experiment with Anolis distichus populations exhibiting different dewlap phenotypes, we raise F1 offspring in a common garden experiment to assess whether dewlap color is inherited. We follow this with carotenoid supplementation to investigate the influence of dietary pigments to dewlap color variation. We find significant differences in several aspects of dewlap color and pattern to persist to the F1 generation (fathers: N = 19; F1 males: N = 50; P < 0.01) with no change in dewlap phenotype with carotenoid supplementation (N = 52; P > 0.05). These results strongly support that genetic differences underlie dewlap color variation, thereby satisfying a key requirement of natural selection. Our findings provide an important stepping-stone to understanding the evolution of an incredibly diverse signal important for sexual selection and species recognition.

Iridophores and not carotenoids account for chromatic variation of carotenoid-based coloration in common lizards (Lacerta vivipara).

Abstract Carotenoids typically need reflective background components to shine. Such components, iridophores, leucophores, and keratin- and collagen-derived structures, are generally assumed to show no or little environmental variability. Here, we investigate the origin of environmentally induced variation in the carotenoid-based ventral coloration of male common lizards (Lacerta vivipara) by investigating the effects of dietary carotenoids and corticosterone on both carotenoid- and background-related reflectance. We observed a general negative chromatic change that was prevented by ß-carotene supplementation. However, chromatic changes did not result from changes in carotenoid-related reflectance or skin carotenoid content but from changes in background-related reflectance that may have been mediated by vitamin A1. An in vitro experiment showed that the encountered chromatic changes most likely resulted from changes in iridophore reflectance. Our findings demonstrate that chromatic variation in carotenoid-based ornaments may not exclusively reflect differences in integumentary carotenoid content and, hence, in qualities linked to carotenoid deposition (e.g., foraging ability, immune response, or antioxidant capacity). Moreover, skin carotenoid content and carotenoid-related reflectance were related to male color polymorphism, suggesting that carotenoid-based coloration of male common lizards is a multicomponent signal, with iridophores reflecting environmental conditions and carotenoids reflecting genetically based color morphs.

Interrelationships between spontaneous and low-level stimulus-frequency otoacoustic emissions in humans.

It has been proposed that OAEs be classified not on the basis of the stimuli used to evoke them, but on the mechanisms that produce them (Shera and Guinan, 1999). One branch of this taxonomy focuses on a coherent reflection model and explicitly describes interrelationships between spontaneous emissions (SOAEs) and stimulus-frequency emissions (SFOAEs). The present study empirically examines SOAEs and SFOAEs from individual ears within the context of model predictions, using a low stimulus level (20 dB SPL) to evoke SFOAEs. Emissions were recorded from ears of normal-hearing young adults, both with and without prominent SOAE activity. When spontaneous activity was observed, SFOAEs demonstrated a localized increase about the SOAE peaks. The converse was not necessarily true though, i.e., robust SFOAEs could be measured where no SOAE peaks were observed. There was no significant difference in SFOAE phase-gradient delays between those with and without observable SOAE activity. However, delays were larger for a 20 dB SPL stimulus level than those previously reported for 40 dB SPL. The total amount of SFOAE phase accumulation occurring between adjacent SOAE peaks tended to cluster about an integral number of cycles. Overall, the present data appear congruous with predictions stemming from the coherent reflection model and support the notion that such comparisons ideally are made with emissions evoked using relatively lower stimulus levels.

Histopathological effects of carbaryl on testes of snake-eyed lizard, Ophisops elegans.

BACKGROUND, AIM AND SCOPE: Due to their ecological niche and insectivore nature, lizards are of increased risk of exposure to pesticides in agricultural areas. In addition to their potential direct effects on non-target species, insecticides can also result in indirect impacts on lizard population by reducing their food source. Carbaryl is a common insecticide that is widely used in areas of Turkey that are home to a variety of reptiles. However, to date, little is known about the potential effects of the exposure of reptiles such as lizards or snakes to this pesticide. The aim of the study was to investigate toxic effects of carbaryl on the testes of snake-eyed lizard, Ophisops elegans that is common to regions in Turkey where Carbaryl is applied and that it can be easily cultured in the laboratory. MATERIAL AND METHODS: Adult male lizards were exposed to carbaryl once by oral gavage in concentrations of 2.5, 25 and 250 µg/g. After 96 h, lizards were euthanized and dissected. Histopathological changes were detected by randomly counting 100 tubules in each lizard. Seminiferous tubules were categorized as normal, sloughing and disorganized tubules. Diameters of tubules were also measured. The differences in histopathological changes and tubule diameters were compared for statistical significance by one-way ANOVA, using SPSS 16.0. RESULTS AND DISCUSSION: Histopathological changes were more prominent in medium- (25 µg/g) and high-dose (250 µg/g) groups than in the low-dose (2.5 µg/g) group. In the medium-dose group, the hexagonal appearance of most tubules disappeared, and they took on an oval shape. Sloughing was the characteristic tubule appearance of the medium group. In the high-dose group, significant increases in the number of disorganized tubules and prominence of haemorrhages was observed. CONCLUSION: Carbaryl caused histopathological defects on the testes of O. elegans, so it is clear that carbaryl affects male fertility in O. elegans.

Superhydrophobic gecko feet with high adhesive forces towards water and their bio-inspired materials.

Functional integration is an inherent characteristic for multiscale structures of biological materials. In this contribution, we first investigate the liquid-solid adhesive forces between water droplets and superhydrophobic gecko feet using a high-sensitivity micro-electromechanical balance system. It was found, in addition to the well-known solid-solid adhesion, the gecko foot, with a multiscale structure, possesses both superhydrophobic functionality and a high adhesive force towards water. The origin of the high adhesive forces of gecko feet to water could be attributed to the high density nanopillars that contact the water. Inspired by this, polyimide films with gecko-like multiscale structures were constructed by using anodic aluminum oxide templates, exhibiting superhydrophobicity and a strong adhesive force towards water. The static water contact angle is larger than 150° and the adhesive force to water is about 66 µN. The resultant gecko-inspired polyimide film can be used as a "mechanical hand" to snatch micro-liter liquids. We expect this work will provide the inspiration to reveal the mechanism of the high-adhesive superhydrophobic of geckos and extend the practical applications of polyimide materials.

Evidence of hepatotoxicity in the sand lizard Acanthodactylus scutellatus from Kuwait's Greater Al-Burgan oil field.

The purpose of this study was to investigate the impact of oil pollution in a desert location (the Greater Al-Burgan oil fields, an area damaged in the Gulf War in 1991) in Kuwait on the hepatotoxicity of the Sand lizard Acanthodactylus scutellatus (A. scutellatus). Twenty lizards (10 of each sex) from each polluted and each control sites were collected. Livers were removed from dissected animals and ready for fixation by Bouin's solution and formal-saline. Twenty sections (10 from males and 10 from females) from each tar mat (polluted) and control sites were prepared and examined for cell diameter and nuclear measurements using Cell Analysis Systems. The cytology of hepatocytes showed normal appearance in samples from the control sites. Dead cells were abundant in the sections of lizard livers from the tar mat sites and occurred in notably greater numbers than the sections of livers of animals from the control sites. Examinations of the data confirm that the cell and nuclear diameters in liver samples of males collected from polluted sites were generally greater than those of corresponding females. The liver sections obtained from animals in the tar mat site had greater cellular diameters than counterparts from control sites. Females from the polluted sites were also affected by oil pollution by having larger hepatocyte diameters and their nuclei were also affected, being larger than female nuclei from the control sites. The most remarkable feature observed in hepatocytes of lizards collected from the tar mat sites were swelling of hepatocytes, ballooning degeneration of hepatic cytoplasm and cell death. This study confirmed that the prolonged exposure to oil pollution may result in increased accumulation of contaminants and may cause severe liver pathology in a range of wild organisms such as A. scutellatus.

Fabrication and analysis of gecko-inspired hierarchical polymer nanosetae.

A gecko's superb ability to adhere to surfaces is widely credited to the large attachment area of the hierarchical and fibrillar structure on its feet. The combination of these two features provides the necessary compliance for the gecko toe-pad to effectively engage a high percentage of the spatulae at each step to any kind of surface topography. With the use of multi-tiered porous anodic alumina template and capillary force assisted nanoimprinting, we have successfully fabricated a gecko-inspired hierarchical topography of branched nanopillars on a stiff polymer. We also demonstrated that the hierarchical topography improved the shear adhesion force over a topography of linear structures by 150%. A systematic analysis to understand the phenomenon was performed. It was determined that the effective stiffness of the hierarchical branched structure was lower than that of the linear structure. The reduction in effective stiffness favored a more efficient bending of the branched topography and a better compliance to a test surface, hence resulting in a higher area of residual deformation. As the area of residual deformation increased, the shear adhesion force emulated. The branched pillar topography also showed a marked increase in hydrophobicity, which is an essential property in the practical applications of these structures for good self-cleaning in dry adhesion conditions.