A simplified molecular method for distinguishing among species and ploidy levels in European water frogs (Pelophylax)

Western Palearctic water frogs in the genus Pelophylax are a set of morphologically similar anuran species that form hybridogenetic complexes. Fully reliable identification of species and especially of hybrid ploidy depends on karyological and molecular methods. In central Europe, native water frog populations consist of the Pelophylax esculentus complex, that is, P. lessonae (LL), P. ridibundus (RR) and the hybrid form P. esculentus that can have different karyotypes (RL, LLR and RRL). We developed existing molecular methods further and propose a simple PCR method based on size‐differences in the length of the serum albumin intron‐1 and the RanaCR1, a non‐LTR retrotransposon of the chicken repeat (CR) family. This PCR yields taxon‐specific banding patterns that can easily be screened by standard agarose gel electrophoresis and correctly identify species in all of the 160 samples that had been identified to karyotype with other methods. To distinguish ploidy levels in LR, LLR and RRL specimens, we used the ratio of the peak heights of the larger (ridibundus specific) to the smaller (lessonae specific) bands of fluorescently labelled PCR products resolved on a capillary DNA sequencer and obtained a correct assignment of the karyotype in 93% of cases. Our new method will cut down time and expenses drastically for a reliable identification of water frogs of the P. esculentus complex and potentially for identification of other hybridogenetic complexes and/or taxa, and it even serves as a good indicator of the ploidy status of hybrid individuals.     

Mode of hybridogenesis and habitat preferences influence population composition of water frogs (Pelophylax esculentus complex,Anura: Ranidae) in a region of sympatric occurrence (western Slovakia)

Coexistence of sperm‐dependent asexual hybrids with their sexual progenitors depends on genetic and ecological interactions between sexual and asexual forms. In this study, we investigate genotypic composition, modes of hybridogenetic gametogenesis and habitat preferences of European water frogs (Pelophylax esculentus complex) in a region of sympatric occurrence. Pelophylax esculentus complex comprises parental species P. ridibundus and P. lessonae, whose primary hybridization leads to hybridogenetic lineages of P. esculentus. Hybrids clonally transmit one parental genome and mate with the other parental species, forming a new generation of hybrids. In the region of western Slovakia, we found syntopic occurrence of diploid and triploid hybrids with P. lessonae, syntopic occurrence of all three taxa as well as the existence of pure P. ridibundus populations. All triploid hybrids were exclusively male possessing one ridibundus and two different lessonae genomes (RLL). Sex ratio in diploid hybrids was substantially female‐biased. Irrespective of the population composition, diploid hybrids excluded the lessonae genome from their germ line and produced ridibundus gametes. Contrarily, RLL males unequivocally eliminated the ridibundus genome and produced diploid lessonae sperms. Perpetuation of RLL males in studied populations is most likely achieved by their mating with diploid hybrid females. The composition of water frog populations is also shaped by taxon‐specific habitat preferences. While P. ridibundus preferred larger water bodies (gravelpits, fishery ponds, dead river arms), P. lessonae was most frequently found in marshes and smaller sandpits. Pelophylax esculentus occupied predominately similar habitats as its sexual host P. lessonae.     

Gamete production patterns,ploidy, and population genetics reveal evolutionary significant units in hybrid water frogs (Pelophylax esculentus)

The European water frog Pelophylax esculentus is a natural hybrid between P. lessonae (genotype LL) and P. ridibundus (RR). It reproduces through hybridogenesis, eliminating one parental genome from its germline and producing gametes containing the genome of the other parental species. According to previous studies, this elimination and transmission pattern is very diverse. In mixed populations, where only diploid hybrids (LR) live in sympatry and mate with one or both parental species, the excluded genome varies among regions, and the remaining genome is transmitted clonally to haploid gametes. In all‐hybrid populations consisting of diploid (LR) and triploid (LLR and/or LRR) frogs, diploid individuals also produce gametes clonally (1n in males, 2n in females), whereas triploids eliminate the genome they have in single copy and produce haploid gametes containing the recombined other genome. However, here, too, regional differences seem to exist, and some triploids have been reported to produce diploid gametes. In order to systematically study such regional and genotype differences in gamete production, their potential origin, and their consequences for the breeding system, we sampled frogs from five populations in three European countries, performed crossing experiments, and investigated the genetic variation through microsatellite analysis. For four populations, one in Poland, two in Germany, and one in Slovakia, our results confirmed the elimination and transmission pattern described above. In one Slovakian population, however, we found a totally different pattern. Here, triploid males (LLR) produce sperm with a clonally transmitted diploid LL genome, rather than a haploid recombined L genome, and LR females clonally produce haploid R eggs, rather than diploid LR eggs. These differences among the populations in gamete production go along with differences in genomotype composition, breeding system (i.e., the way triploids are produced), and genetic variation. These differences are strong evidence for a polyphyletic origin of triploids. Moreover, our findings shed light on the evolutionary potential inherent to the P. esculentus complex, where rare events due to untypical gametogenetic processes can lead to the raise, the perpetuation, and the dispersion of new evolutionary significant lineages which may also deserve special conservation measures.     

Development and growth of long bones in European water frogs (Amphibia: Anura: Ranidae), with remarks on age determination

Differentiation and development of long bones were studied in European water frogs: Rana lessonae, R. ridibunda, and R. esculenta. The study included premetamorphic larvae (Gosner Stage 40) to frogs that were 5 years old. Femora, metatarsal bones, and proximal phalanges of the hindlimb exhibit the same pattern of periosteal bone differentiation and the same pattern of growth. Longitudinal and radial growth of these bones was studied by examination of the diaphyses and epiphyses, particularly where the edge of periosteal bone is inserted into the epiphysis. The periosteum seems to be responsible for both longitudinal and radial growth. Investigation of the formation, length, and arrangement of lines of arrested growth reveals that the first line is present only in the middle 25-35% of the length of the diaphysis of an adult bone; therefore, only the central portion of the diaphysis should be used for age estimation in skeletochronological studies. Comparison of the shapes and histological structures of epiphyses in the femur, metatarsal bones, and phalanges revealed that epiphyseal cartilages are composed of an inner and outer part. The inner metaphyseal cartilage has distinct zones and plugs the end of the periosteal bone cylinder; its role in longitudinal growth is questioned. The outer epiphyseal cartilage is composed of articular cartilages proper, in addition to lateral articular cartilages. Differences in the symmetry of the lateral articular cartilages of distal epiphyses of the femur and toes may reflect adaptations to different kinds of movements at the knee and in the foot.     

Taxonomic survey of water frog populations of <Emphasis Type="Italic">Pelophylax bedriagae</Emphasis> (Anura: Ranidae) in western Iran: a morphometric and bioacoustic approach

We explored the population systems of Pelophylax bedriagae (Camerano, 1882) in northwestern, western and southwestern Iran. Statistical significance for a hypothesis of sexual dimorphism of the frogs was tested using t-test and multivariate analysis of variances (MANOVA). Due to lack of sexual differences between the male and female frogs, the data of both sexes were pooled for further analysis. Taxonomic relationships among local populations were investigated using discriminate analysis (by stepwise method) with iterative sample assignment. The multivariate morphometric analyses revealed that the range of P. bedriagae extends to western Iran. The next step was to analyze the advertisement call (five standard parameters: call duration, number of pulse groups per call, duration of pulse groups, interval between pulse groups, and intercall interval) of the water frogs inhabiting three sites in western Iran. Rigorous application of the bioacoustic method removes all doubt that these three sites are inhabited by the same species, which on the basis of priority of nomenclature should be called P. bedriagae.     

Confronting assumptions about spontaneous autogamy in populations of Eulophia alta (Orchidaceae) in south Florida: assessing the effect of pollination treatments on seed formation,seed germination and seedling development

The breeding system of the terrestrial orchid Eulophia alta was investigated in south Florida where it has previously been reported as an auto‐pollinated species. The effect of breeding system on seed viability and germinability and seedling development was also investigated. Incidences of spontaneous autogamy in E. alta were rare at the study site, resulting in only 7.1% of observed flowers forming capsules. In addition, hand pollination resulted in significantly greater capsule formation when flowers were subjected to induced autogamy (46.4%), artificial geitonogamy (64.3%) and xenogamy at both short (pollen source 10–100 m away; 42.9%) and long (pollen source > 10 km away; 67.9%) distances. Pollen source had little effect on seed viability and germinability or seedling growth rates. However, seed resulting from spontaneous autogamy developed more slowly than seed originating from the other treatments. These data indicate that spontaneous autogamy is rare in E. alta and that naturally forming capsules may be the result of unobserved pollination events. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161 , 78–88.     

Neighbouring‐group composition and within‐group relatedness drive extra‐group paternity rate in the European badger (Meles meles)

Extra‐group paternity (EGP) occurs commonly among group‐living mammals and plays an important role in mating systems and the dynamics of sexual selection; however, socio‐ecological and genetic correlates of EGP have been underexplored. We use 23 years of demographic and genetic data from a high‐density European badger (Meles meles) population, to investigate the relationship between the rate of EGP in litters and mate availability, mate incompatibility and mate quality (heterozygosity). Relatedness between within‐group assigned mothers and candidate fathers had a negative quadratic effect on EGP, whereas the number of neighbouring‐group candidate fathers had a linear positive effect. We detected no effect of mean or maximum heterozygosity of within‐group candidate fathers on EGP. Consequently, EGP was associated primarily with mate availability, subject to within‐group genetic effects, potentially to mitigate mate incompatibility and inbreeding. In badgers, cryptic female choice, facilitated by superfecundation, superfoetation and delayed implantation, prevents males from monopolizing within‐group females. This resonates with a meta‐analysis in group‐living mammals, which proposed that higher rates of EGP occur when within‐group males cannot monopolize within‐group females. In contrast to the positive meta‐analytic association, however, we found that EGP associated negatively with the number of within‐group assigned mothers and the number of within‐group candidate fathers; potentially a strategy to counter within‐group males committing infanticide. The relationship between the rate of EGP and socio‐ecological or genetic factors can therefore be intricate, and the potential for cryptic female choice must be accounted for in comparative studies.     

A decade of proteomics accomplished! Central and Eastern European Proteomic Conference (CEEPC) celebrates its 10th Anniversary in Budapest,Hungary

The Central and Eastern European Proteomic Conference (CEEPC) proudly celebrated its 10th Anniversary with an exciting scientific program inclusive of proteome, proteomics and systems biology in Budapest, Hungary. Since 2007, CEEPC has represented ‘state-of the-art’ proteomics in and around Central and Eastern Europe and these series of conferences have become a well-recognized event in the proteomic calendar. Fresher challenges and global healthcare issues such as ageing and chronic diseases are driving clinical and scientific research towards regenerative, reparative and personalized medicine. To this end, proteomics may enable diverse intertwining research fields to reach their end goals. CEEPC will endeavor to facilitate these goals.     

Multigeneration Reproductive Study of Hydroxyprogesterone Caproate (HPC) in the Rat: Laboratory Results and Clinical Significance

To demonstrate reproductive safety of a new commercial product for reducing the risk of preterm birth, HPC (17α‐hydroxyprogesterone caproate, Makena; manufactured by Baxter Pharmaceutical Solutions, Bloomington IN for Ther‐Rx Corporation, St. Louis, MO) was administered intramuscularly in Charles River LaboratoryCD strain rats. HPC was given at intervals equal to the half‐life measured in rats during three phases of embryo‐fetal development: during the period of ovarian development (RP1, days 8, 14, and 20), following implantation of the embryo (TP, days 6, 12, and 18), and, corresponding to the start of the drug in week 16 or later in humans, after gonadal formation including differentiation of the testes (RP2, day 17). Dose levels up to 30× the human therapeutic doses were utilized including 0 (vehicle), 5, 25, and 150 mg/kg (volume 0.6 ml/kg). Four groups of 25 time‐mated rats each were used for each phase. In addition, equal numbers of naïve (untreated) rats of opposite gender were used for F₁ breeding studies. HPC did not produce any consistent test‐article–related findings in the treated F₀ dams, their developing F₁ fetuses and did not affect the ability of the latter to produce a viable F₂ generation. The F₁ offspring did not evidence any adverse effects during their behavioral, sensory, and developmental assessments, including teratogenicity. Based on the cumulative data obtained from rats treated over two generations and during development in this study, the No‐observable‐effect‐level (NOEL) was established as 150 mg/kg. This study supports the absence of reproductive toxicity with HPC in published studies in animal models and in human clinical trials.     

Isolation and characterization of microsatellites in two tropical butterflies,Drupadia theda and Arhopala epimuta (Lepidoptera: Lycaenidae)

Little is known about the effect of El Niño Southern Oscillation‐induced fires on the genetic diversity of tropical rainforest species. Here, I report on the isolation and characterization of 10 microsatellite loci, five loci each, for two lycaenid butterfly species in East Kalimantan, Indonesia, namely Drupadia theda and Arhopala epimuta, which will be used to specifically study the impact of disturbance on genetic diversity. Microsatellite enrichment was carried out using streptavidin‐coated magnetic beads. Positive colonies were identified with the three‐primer polymerase chain reaction (PIMA). Cross‐species amplifications conducted both within and between genera were successful in 16 out of 20 tests.     

A diabetes-like environment increases malformation rate and diminishes prostaglandin E(2) in rat embryos: reversal by administration of vitamin E and folic acid

BACKGROUND: Offspring of women with diabetes are at increased risk for congenital malformations and disturbed growth compared with infants from nondiabetic pregnancies. The precise biological process behind these effects is not yet completely clarified. Previous studies have suggested that diabetic embryopathy is associated with increased level of oxidative stress and disturbed arachidonic acid metabolism. The aim of the present study was to investigate whether a diabetes-like environment both in vivo and in vitro increases embryonic levels of isoprostanes and alters embryonic prostaglandin E(2) (PGE(2)) concentration. Furthermore, we studied whether vitamin E and folic acid treatment rectify such alterations. METHODS: Embryos from diabetic and nondiabetic rats at gestational days (GDs) 10 and 11 were used. In the in vitro experiments, we used whole embryo culture, which mimics pregnancy. GD 9 embryos from nondiabetic rats were cultured for either 24 hr (corresponding to GD 10) or 48 hr (corresponding to GD 11) and exposed to 10 or 30 mM glucose concentration with or without folic acid. RESULTS: Embryos from diabetic rats and embryos cultured in a high glucose concentration showed increased malformation rates. Dietary treatment with vitamin E in vivo and supplementation of folic acid in the culture medium with 30 mM glucose in vitro decreased the malformation rate, decreased embryonic isoprostane levels, and increased PGE(2) concentration. CONCLUSIONS: Diabetes-induced oxidative stress and disturbance of PGE(2) production may contribute to the embryonic dysmorphogenesis in the offspring of diabetic rodents and, thereby, may also have a role in human diabetic embryopathy.     

Poly(ADP-ribose) turnover in quail myoblast cells: Relation between the polymer level and its catabolism by glycohydrolase

The concerted action of poly(ADP-ribose) polymerase (PARP) which synthesizes the poly(ADP-ribose) (pADPr) in response to DNA strand breaks and the catabolic enzyme poly(ADP-ribose) glycohydrolase (PARG) determine the level of polymer and the rate of its turnover. In the present study, we have shown that the quail myoblast cells have high levels of basal polymer as compared to the murine C3H10T1/2 fibroblasts. We have conducted this study to investigate how such differences influence polymer synthesis and its catabolism in the cells in response to DNA damage by alkylating agent. In quail myoblast cells, the presence of high MNNG concentration such as 200 \sgmaelig;M for 30 min induced a marginal decrease of 15% in the NAD content. For C3H10T1/2 cell line, 64 \sgmaelig;M MNNG provoked a depletion of NAD content by approximately 50%. The induction of the polymer synthesis in response to MNNG treatment was 6-fold higher in C3H10T1/2 cells than in quail myoblast cells notwithstanding the fact that 3-fold higher MNNG concentration was used for quail cells. The polymer synthesis thus induced in quail myoblast cells had a 4-5 fold longer half life than those induced in C3H10T1/2 cells. To account for the slow turnover of the polymer in the quail myoblast cells, we compared the activities of the polymer catabolizing enzyme (PARG) in the two cell types. The quail myoblast cells had about 25% less activity of PARG than the murine cells. This difference in activity is not sufficient to explain the large difference of the rate of catabolism between the two cell types implicating other cellular mechanisms in the regulation of pADPr turnover.     

Diet composition of the endangered giant water bug Lethocerus deyrolli (Hemiptera: Belostomatidae) in the rice fields of Japan: Which is the most important prey item among frogs,fish, and aquatic insects?

The food habits of the endangered giant water bug, Lethocerus deyrolli, were studied in the rice fields of Nose, in the north of Osaka Prefecture, Japan. Field observations revealed that frogs were the most important prey item. Frogs represented 86.4% and 78.6% in the diet of L. deyrolli in spring and summer, respectively. Among seven species of three families (Hylidae, Rhacophoridae, and Ranidae) exploited by L. deyrolli, the most important food item was adult Hyla japonica in spring and juvenile Rana nigromaculta in summer. Fish and aquatic arthropods were not considered important foods for L. deyrolli. The frog‐dependent food habits indicate that the recovery and conservation of frogs should be prioritized to protect L. deyrolli from extinction.     

Determination of alpha-helix and beta-sheet stability in the solid state: a solid-state NMR investigation of poly(L-alanine)

The relative stability of alpha-helix and beta-sheet secondary structure in the solid state was investigated using poly(L-alanine) (PLA) as a model system. Protein folding and stability has been well studied in solution, but little is known about solid-state environments, such as the core of a folded protein, where peptide packing interactions are the dominant factor in determining structural stability. (13)C cross-polarization with magic angle spinning (CPMAS) NMR spectroscopy was used to determine the backbone conformation of solid powder samples of 15-kDa and 21.4-kDa PLA before and after various sample treatments. Reprecipitation from helix-inducing solvents traps the alpha-helical conformation of PLA, although the method of reprecipitation also affects the conformational distribution. Grinding converts the secondary structure of PLA to a final steady-state mixture of 55% beta-sheet and 45% alpha-helix at room temperature regardless of the initial secondary structure. Grinding PLA at liquid nitrogen temperatures leads to a similar steady-state mixture with 60% beta-sheet and 40% alpha-helix, indicating that mechanical shear force is sufficient to induce secondary structure interconversion. Cooling the sample in liquid nitrogen or subjecting it to high pressure has no effect on secondary structure. Heating the sample without grinding results in equilibration of secondary structure to 50% alpha-helix/50% beta-sheet at 100 degrees C when starting from a mostly alpha-helical state. No change was observed upon heating a beta-sheet sample, perhaps due to kinetic effects and the different heating rate used in the experiments. These results are consistent with beta-sheet approximately 260 J/mol more stable than alpha-helix in solid-state PLA.     

Cryptic diversity and species delimitation in the Xiphinema americanum‐group complex (Nematoda: Longidoridae) as inferred from morphometrics and molecular markers

The Xiphinema americanum‐group constitutes a complex of about 55 species of polyphagous plant‐ectoparasitic nematodes with a worldwide distribution. This group of plant‐parasitic nematodes is one of the most difficult dagger nematode species complexes for diagnosis because the morphology is very conservative and morphometric characters often overlap. We conducted nematode surveys in cultivated and wild olives in southern Spain from 2012 to 2014, from which we identified 16 nematode populations of the X. americanum‐group, five of which were tentatively identified as belonging to three new species and are described herein as X iphinema plesiopachtaicum sp. nov., X iphinema vallense sp. nov. , and X iphinema astaregiense sp. nov. , and 11 populations belonging to nine known species: Xiphinema brevisicum, Xiphinema duriense, Xiphinema incertum, Xiphinema luci, Xiphinema madeirense, Xiphinema opisthohysterum, Xiphinema pachtaicum, Xiphinema parapachydermum, and Xiphinema rivesi. A phenetic study based on multivariate factor analyses was developed to compare some of these related species by using morphometric features. In the factor analysis the first four factors accounted for 73.1% of the total variance of the selected characters, identifying body length, body length/maximum body width (a), body length/pharyngeal length (b), body length/tail length (c), and tail length/body width at anus (c′) ratios, distance from anterior end to vulva as percentage of body length (V), stylet length, oral aperture‐guiding ring distance, and lip region width as key morphometric characters to differentiate a restricted set of species within the X. pachtaicum‐subgroup that includes X. plesiopachtaicum sp. nov. and X. vallense sp. nov. Multivariate analysis of variance using these specific characters allowed to differentiate species in the X. pachtaicum complex or groups of them using morphometric characters (body length, a, b, c, c′, V, stylet length, lip region width, oral aperture‐guiding ring distance, female tail length, and hyaline region length). Phylogenetic analyses based on nuclear ribosomal DNA genes [D2‐D3 expansion segments of large ribosomal subunit 28S, and internal transcribed spacer 1 (ITS1)] and the protein‐coding mitochondrial gene, cytochrome c oxidase subunit 1 (coxI) were congruent, showing two main clades separating most of the species of X. americanum‐subgroup ‘sensu stricto’ from the X. pachtaicum‐subgroup. Agreement between phylogenetic trees and some morphological characters (viz. total stylet length, vulva position, and a ratio) were tested by reconstruction of their histories on rRNA‐based trees using parsimony and Bayesian approaches. Thus, integrative taxonomy, based on a combination of multivariate morphological and molecular analyses constitutes a new insight into the identification of X. americanum‐group species. © 2015 The Linnean Society of London     

Rhipicephalus (Boophilus) microplus: distinct acute phase proteins vary during infestations according to the genetic composition of the bovine hosts, Bos taurus and Bos indicus

Tick bites may trigger acute phase responses. Positive and negative acute phase proteins were measured in infested cattle genetically resistant and susceptible to ticks. During heavier infestations levels of haptoglobin increased significantly in susceptible bovines; levels of serum amyloid A increased in resistant bovines; levels of alpha-1-acid glycoprotein decreased significantly in resistant bovines; levels of transferrin decreased significantly in susceptible bovines. In conclusion, tick infestations trigger acute phase responses and enhancement of specific acute phase proteins differs according to the genetic composition of hosts. Acute phase proteins may constitute useful biological signatures for monitoring the stress induced by tick infestations.     

Myristoylation of human LanC-like Protein 2 (LANCL2) is essential for the interaction with the plasma membrane and the increase in cellular sensitivity to adriamycin

Human LANCL2, also known as Testis-specific Adriamycin Sensitivity Protein (TASP), is a member of the highly conserved and widely distributed lanthionine synthetase component C-like (LANCL) protein family. Expression studies of tagged LANCL2 revealed the major localization to the plasma membrane, juxta-nuclear vesicles, and the nucleus, in contrast to the homologue LANCL1 that was mainly found in the cytosol and nucleus. We identified the unique N-terminus of LANCL2 to function as the membrane anchor and characterized the relevant N-terminal myristoylation and a basic phosphatidylinositol phosphate-binding site. Interestingly, the non-myristoylated protein was confined to the nucleus indicating that the myristoylation targets LANCL2 to the plasma membrane. Cholesterol depletion by methyl-β-cyclodextrin caused the partial dissociation of overexpressed LANCL2 from the plasma membrane in vitro, whereas in vivo we observed an enhanced cell detachment from the matrix. We found that overexpressed LANCL2 interacts with the cortical actin cytoskeleton and therefore may play a role in cytoskeleton reorganization and in consequence to cell detachment. Moreover, we confirmed previous data that LANCL2 overexpression enhances the cellular sensitivity to the anticancer drug adriamycin and found that this sensitivity is dependent on the myristoylation and membrane association of LANCL2.