A staging table of Balkan crested newt embryonic development to serve as a baseline in evolutionary developmental studies.

There is an increased interest in the evolution and development of newts from the genus Triturus because: (1) morphological differentiation among the nine constituent species largely corresponds to different ecological preferences, (2) hybridization between different species pairs has various evolutionary outcomes in terms of life history traits and morphology, and (3) the genus expresses a balanced lethal system that causes arrested growth and death of half of the embryos. These features provide natural experimental settings for molecular, morphological, and life-history studies. Therefore, we produce a staging table for the Balkan crested newt (T. ivanbureschi). We provide detailed descriptions of 34 embryonic stages based on easily observable and interpretable external morphological characters, to ensure reproducibility. Compared with previous staging tables for Triturus, we include a vastly increased sample size and provide high-resolution photographs in lateral, ventral, and dorsal view, complemented by videos of specific developmental periods, and accompanied by detailed explanations on how to delineate the specific stages. Our staging table will serve as a baseline in comparative studies on Triturus newts: an emerging model system in evolutionary and developmental studies.

Morphological variation, modularity and integration in the scapula and humerus of Lissotriton newts.

The modular organization of tetrapod paired limbs and girdles, influenced by the expression of Hox genes is one of the primary driving forces of the evolution of animal locomotion. The increased morphological diversification of the paired limbs is correlated with reduced between-limb covariation, while correlation within the elements is usually higher than between the elements. The tailed amphibians, such as Lissotriton newts, have a biphasic lifestyle with aquatic and terrestrial environments imposing different constraints on limb skeleton. By employing the methods of computerized microtomography and 3D geometric morphometrics, we explored the pattern of morphological variation, disparity, modularity and morphological integration in the proximal parts of the anterior limbs of six species of Eurasian small bodied newts. Although the species significantly differ in limb shape, there is a great overlap in morphology of scapula and humerus, and there are no differences in morphological disparity. For the scapula, the shape differences related to the duration of the aquatic period are in length, depth and curvature. The shape of the humerus is not affected by the length of aquatic period, and shape differences between the species are related to robustness of the body. The length of aquatic period has statistically supported phylogenetic signal. The scapula and humerus are structures of varying modularity. For the humerus, the strongest support on the phylogenetic level was for the capitulum/shaft hypothesis, which can also be interpreted as functional modularity. For the scapula, the greatest support was for the antero-posterior hypothesis of modularity in case of Lissotriton vulgaris, which can be explained by different functional roles and muscle insertion patterns, while there was no phylogenetic modularity. The modularity patterns seem to correspond with the general tetrapod pattern, with modularity being more pronounced in the distal structure. The future research should include more salamandrid taxa with different habitat preferences and both adult and larval stages, in order to explore how size, phylogeny and ecology affect the morphology and covariation patterns of limbs.

Regionalization and morphological integration in the vertebral column of Eurasian small-bodied newts (Salamandridae: Lissotriton).

Serially homologous structures may have complex patterns of regionalization and morphological integration, influenced by developmental Hox gene expression and functional constraints. The vertebral column, consisting of a number of repeated, developmentally constrained, and highly integrated units-vertebrae-is such a complex serially homologous structure. Functional diversification increases regionalization and modularity of the vertebral column, particularly in mammals. For salamanders, three concepts of regionalization of the vertebral column have been proposed, recognizing one, two, or three presacral regions. Using three-dimensional geometric morphometrics on vertebra models acquired with microcomputerized tomography scanning, we explored the covariation of vertebrae in four closely related taxa of small-bodied newts in the genus Lissotriton. The data were analyzed by segmented linear regression to explore patterns of vertebral regionalization and by a two-block partial least squares method to test for morphological integration. All taxa show a morphological shift posterior to the fifth trunk vertebra, which corresponds to the two-region concept. However, morphological integration is found to be strongest in the mid-trunk. Taken jointly, these results indicate a highly integrated presacral vertebral column with a subtle two-region differentiation. The results are discussed in relation to specific functional requirements, developmental and phylogenetic constraints, and specific requirements posed by a biphasic life cycle and different locomotor modes (swimming vs. walking). Further research should be conducted on different ontogenetic stages and closely related but ecologically differentiated species.

Variation in vertebrae shape across small-bodied newts reveals functional and developmental constraints acting upon the trunk region.

The salamander vertebral column is largely undifferentiated with a series of more or less uniform rib-bearing presacral vertebrae traditionally designated as the trunk region. We explored regionalization of the salamander trunk in seven species and two subspecies of the salamander genus Lissotriton by the combination of microcomputed tomography scanning and geometric morphometrics. The detailed information on trunk vertebral shape was subjected to a multidimensional cluster analysis and a phenotypic trajectory analysis. With these complementary approaches, we observed a clear morphological regionalization. Clustering analysis showed that the anterior trunk vertebrae (T1 and T2) have distinct morphologies that are shared by all taxa, whereas the subsequent, more posterior vertebrae show significant disparity between species. The phenotypic trajectory analysis revealed that all taxa share a common pattern and amount of shape change along the trunk region. Altogether, our results support the hypothesis of a conserved anterior-posterior developmental patterning which can be associated with different functional demands, reflecting (sub)species' and, possibly, regional ecological divergences within species.

Valuation of Informal Care Provided to People Living With Dementia: A Systematic Literature Review.

OBJECTIVES: This study aimed to identify the methods used to determine the value of informal care provided to people living with dementia and to estimate the average hourly unit cost by valuation method. METHODS: A literature search in MEDLINE Complete, CINAHL, PsycINFO, EconLit, EMBASE and NHS Economic Evaluation Database was undertaken. Following the screening of title, abstract, and full text, characteristics of eligible studies were extracted systematically and analyzed descriptively. The corresponding hourly cost estimates were converted into 2018 US dollars based on purchasing power parities for gross domestic product. RESULTS: A total number of 111 articles were included in this review from 3106 post-deduplication records. Three main valuation methodologies were identified: the replacement cost method (n = 50), the opportunity cost approach (n = 36), and the stated preference method based on willingness to pay (n = 3), with 16 studies using multiple methods and 6 studies not specifying the valuation method. The amount of informal care increased as the condition of dementia progressed, which was reflected in the cost of informal care. The average hourly unit cost used to value informal care was US $16.78 (SD = US $12.11). Although the unit cost was approximately US $15 per hour when using the opportunity cost method and US $14 when using the stated preference method, the highest unit cost was obtained when using the replacement cost method (US $18.37, SD = US $13.12). CONCLUSIONS: Although costs of informal care should be considered when undertaking an economic evaluation or estimating the overall costs of dementia from a policy and priority-setting perspective, further research into applying consistent approaches to valuation is warranted.

Developmental histology of the liver in the Balkan crested newt Triturus ivanbureschi (Caudata: Salamandridae).

The liver, a crucial organ for metabolic processes, has a generally uniform histological structure across amphibian taxa. However, ontogenetic changes, particularly those related to biphasic life cycle and metamorphosis, are less documented. Here, we explored and described the liver histology of an emerging model organism, the Balkan crested newt (Triturus ivanbureschi) at three ontogenetic stages: larval, juvenile (just after metamorphosis) and adult. At the larval stage, the liver is characterized by hepatocytes containing large lipid droplets, poorly developed basement membranes in the blood vessels, and a lack of melanin-rich macrophage centers. Juveniles show transitional characteristics between larvae and adults. Lipid droplets in hepatocytes are abundant, but also, at the juvenile stage the well-developed basement membrane of blood vessels and melanomacrophages are present, as in adults. The presence of lipid droplets in hepatocytes during larval and juvenile stages suggests the liver's role in fat storage and energy provision during development and growth. Melanomacrophages, which synthesize melanin, perform phagocytosis, and neutralize free radicals, have been found in juveniles (after metamorphosis) and increase with age. The biphasic life cycle and liver histology transition in Triturus newts provide an insight in changes in liver histology and make them a suitable model for studying fat deposition regulation and the evolution of the immune system in terrestrial vertebrates.

Ladies in armor: A micro-computed tomographic study of skin calcification in European toads (genus Bufo).

Micro-computed tomography is a powerful tool toward the detailed reconstruction of internal and external morphology, in particular for ossified and other dense tissues. Here, we document and compare the level of calcification in the skin of the head and the parotoids (the external skin glands) in males and females of common and spined toads, Bufo bufo and B. spinosus. In some anurans, including Bufo species, a specific acellular calcified tissue layer within the dermis has been documented (the Eberth-Katschenko, or EK-layer). By a combination of micro-computed tomography and classical histology, we detected additional calcium deposits located in the dermal layer stratum spongiosum, positioned above the EK-layer. We showed that the level of calcification and the presence of additional calcium deposits are size and sex related, increasing in the order B. bufo males, B. spinosus males, B. bufo females to B. spinosus females. The last of these groups is the least variable. Bufo spinosus females have dense calcium deposits in the parotoids and the dorsal and ventral skin. Three-dimensional volume renderings and cross-sectional slices obtained by micro-CT scanning indicate that this approach is a promising technique for further studies on bufonid skin anatomy and geographic variation in skin calcification.

Histological changes of the skin during postembryonic development of the crested newt Triturus ivanbureschi (Urodela, Salamandridae).

BACKGROUND: Amphibian skin has been studied for many decades, especially the metamorphic changes in the skin of frogs. Less attention has been paid to salamander skin. Here, we describe changes in the skin structure during postembryonic development in a salamandrid species, the Balkan crested newt Triturus ivanbureschi. METHOD: Using traditional histological techniques we examined the skin in the trunk region of three premetamorphic larval stages (hatchling, mid larval and late larval) and two postmetamorphic stages (juvenile, just after metamorphosis, and adult). RESULTS: In larval stages, skin consists only of the epidermis, which gradually develops from the single epithelial cell layer in hatchlings, to a stratified epidermis with gland nests and characteristic Leydig cells at the late larval stage. During metamorphosis, Leydig cells disappear, and the dermal layer develops. In postmetamorphic stages, skin is differentiated on stratified epidermis and the dermis with well-developed glands. Three types of glands were observed in the skin of the postmetamorphic stages: mucous, granular and mixed. Gland composition appears to be stage- and sex-specific, with juveniles and adult female being more similar to each other. In juveniles and adult female, there are a similar proportion of glands in both dorsal and ventral skin, whereas in adult male granular glands dominated the dorsal skin, while mixed glands dominated the ventral skin. CONCLUSION: Our results provide a baseline for future comparative research of skin anatomy in salamanders.

Changes in thyroid histomorphology and thyroglobulin immunostaining upon exposure to thiourea in Triturus newts.

Amphibians are useful bioindicators for monitoring aquatic health and the influence of xenobiotics such as endocrine disrupting chemicals. Because aquatic ecosystems experience the majority of global pollution, aquatic organisms are most exposed and vulnerable to endocrine disruptors. Furthermore, penetration of endocrine disruptors into aquatic organisms especially in amphibians is even easier because of more permeable skin, resulting in high bioavailability and bioaccumulation of chemicals. One of the most potent endocrine disruptors is thiourea, which chemically blocks the synthesis of thyroid hormones and prevents metamorphosis in amphibians. We investigated the influence of thiourea on histomorphology of the thyroid gland in Triturus newts at the metamorphic stage, when thyroid hormone concentrations should reach their maximum level. Chronic exposure to thiourea induced hypertrophy and hyperplasia of follicular cells as well as a significant reduction of interstitial tissue. The intensity of the thyroglobulin immunostaining signal significantly decreases upon chronic exposure to thiourea. Successful cross-reactivity of human primary antibody in immunochemical detection of thyroglobulin in Urodela confirms potential homology in thyroglobulin structure throughout the vertebrates.

The Reproductive Success of Triturus ivanbureschi × T. macedonicus F1 Hybrid Females (Amphibia: Salamandridae).

Two large-bodied newt species, Triturus ivanbureschi and T. macedonicus, hybridize in nature across the Balkan Peninsula. Consequences of hybridization upon secondary contact of two species include species displacement and asymmetrical introgression of T. ivanbureschi mtDNA. We set an experimental reciprocal cross of parental species and obtained two genotypes of F1 hybrids (with T. ivanbureschi or T. macedonicus mtDNA). When hybrids attained sexual maturity, they were engaged in mutual crossings and backcrossing with parental species. We followed reproductive traits over two successive years. Our main aim was to explore the reproductive success of F1 females carrying different parental mtDNA. Additionally, we tested for differences in reproductive success within female genotypes depending on the crossing with various male genotypes (hybrids or parental species). Both female genotypes had similar oviposition periods, number of laid eggs and hatched larvae but different body and egg sizes. Overall reproductive success (percentage of egg-laying females and viability of embryos) was similar for both genotypes. The type of crossing led to some differences in reproductive success within female genotypes. The obtained results suggest that processes that led to exclusion of T. macedonicus mtDNA in natural populations may be related to the survival at postembryonic stages of F2 generation or reproductive barriers that emerged in subsequent hybrid generations.

Effects of thiourea on the skull of Triturus newts during ontogeny.

BACKGROUND: In amphibians, thyroid hormone (TH) has a profound role in cranial development, especially in ossification of the late-appearing bones and remodeling of the skull. In the present study, we explored the influence of TH deficiency on bone ossification and resulting skull shape during the ontogeny of Triturus newt hybrid larvae obtained from interspecific crosses between T. ivanbureschi and T. macedonicus. METHODS: Larvae were treated with two concentrations of thiourea (an endocrine disruptor that chemically inhibits synthesis of TH) during the midlarval and late larval periods. Morphological differences of the cranium were assessed at the end of the midlarval period (ontogenetic stage 62) and the metamorphic stage after treatment during the late larval period. RESULTS: There was no difference in the ossification level and shape of the skull between the experimental groups (control and two treatment concentrations) at stage 62. During the late larval period and metamorphosis, TH deficit had a significant impact on the level of bone ossification and skull shape with no differences between the two treatment concentrations of thiourea. The most pronounced differences in bone development were: the palatopterygoid failed to disintegrate into the palatal and pterygoid portions, retardation was observed in development of the maxilla, nasal and prefrontal bones and larval organization of the vomer was retained in thiourea-treated larvae. CONCLUSIONS: This implies that deficiency of TH caused retardation in development and arrested metamorphic cranium skeletal reorganization, which resulted in divergent cranial shape compared to the control group. Our results confirmed that skull remodeling and ossification of late-appearing bones is TH-dependent, as in other studied Urodela species. Also, our results indicate that TH plays an important role in the establishment of skull shape during the ontogeny of Triturus newts, especially during the late larval period and metamorphosis, when TH concentrations reach their maximum.

Oxidative Stress Parameters in Goitrogen-Exposed Crested Newt Larvae (Triturus spp.): Arrested Metamorphosis.

Thiourea is an established disruptor of thyroid hormone synthesis and is frequently used as an inhibitor of metamorphosis. The changes caused by thiourea can affect processes associated with the oxidative status of individuals (metabolic rate, the HPI axis, antioxidant system). We investigated the parameters of oxidative stress in crested newt (Triturus spp.) larvae during normal development in late larval stage 62 and newly metamorphosed individuals, and during thiourea-stimulated metamorphosis arrest in individuals exposed to low (0.05%) and high (0.1%) concentrations of thiourea. Both groups of crested newts exposed to thiourea retained their larval characteristics until the end of the experiment. The low activities of antioxidant enzymes and the high lipid peroxidation level pointed to increased oxidative stress in larvae at the beginning of stage 62 as compared to fully metamorphosed individuals. The activities of catalase (CAT) and glutathione-S-transferase (GST) and the concentration of sulfhydryl (SH) groups were significantly lower in larvae reared in aqueous solutions containing thiourea than in newly metamorphosed individuals. The high thiourea concentration (0.1%) affected the antioxidative parameters to the extent that oxidative damage could not be avoided, contrary to a lower concentration. Our results provide a first insight into the physiological adaptations of crested newts during normal development and simulated metamorphosis arrest.

Absence of heterosis in hybrid crested newts.

Relationships between phylogenetic relatedness, hybrid zone spatial structure, the amount of interspecific gene flow and population demography were investigated, with the newt genus Triturus as a model system. In earlier work, a bimodal hybrid zone of two distantly related species combined low interspecific gene flow with hybrid sterility and heterosis was documented. Apart from that, a suite of unimodal hybrid zones in closely related Triturus showed more or less extensive introgressive hybridization with no evidence for heterosis. We here report on population demography and interspecific gene flow in two Triturus species (T. macedonicus and T. ivanbureschi in Serbia). These are two that are moderately related, engage in a heterogeneous uni-/bimodal hybrid zone and hence represent an intermediate situation. This study used 13 diagnostic nuclear genetic markers in a population at the species contact zone. This showed that all individuals were hybrids, with no parentals detected. Age, size and longevity and the estimated growth curves are not exceeding that of the parental species, so that we conclude the absence of heterosis in T. macedonicus-T. ivanbureschi. Observations across the genus support the hypothesis that fertile hybrids allocate resources to reproduction and infertile hybrids allocate resources to growth. Several Triturus species hybrid zones not yet studied allow the testing of this hypothesis.

Pharmacist elicited medication histories in the Emergency Department: Identifying patient groups at risk of medication misadventure.

UNLABELLED: The Australian Pharmaceutical Advisory Committee guidelines call for a detailed medication history to be taken at the first point of admission to an Emergency Department (ED). The elderly, in particular those residing in Residential Aged Care Facilities and those with a non-English speaking background, have been identified as patient groups vulnerable to medication misadventure. OBJECTIVE: to analyse the incidence of discrepancies in medication histories in these demographic groups when pharmacist elicited medication histories were compared with those taken by ED physicians. It also aimed to investigate the incidence of medication related ED presentations. METHODS: The study was conducted over a six week period and included 100 patients over the age of 70, who take five or more regular medications, have three or more clinical co-morbidities and/or have been discharged from hospital in three months prior to the study. RESULTS: Twenty four participants were classified as 'language barrier'; 12 participants were from residential aged care facilities, and 64 participants were classified as 'general'. The number of correctly recorded medications was lowest in the 'language barrier' group (13.8%) compared with 18% and 19.6% of medications for 'general' patients and patients from residential aged care facilities respectively. Seven of the patients (29.2%) with 'language barrier'; 1 from a residential aged care facility (8.3%) and 13 of the (20.3%) patients from the 'general' category were suspected as having a medication related ED presentation. CONCLUSION: This study further highlights the positive contribution an ED pharmacist can make to enhancing medication management along the continuum of care. This study also confirms the vulnerability of patients with language barrier to medication misadventure and their need for interpreter services at all stages of their hospitalisation, in particular at the point of ED presentation.