Insertion sequence-caused large-scale rearrangements in the genome of Escherichia coli

A majority of large-scale bacterial genome rearrangements involve mobile genetic elements such as insertion sequence (IS) elements. Here we report novel insertions and excisions of IS elements and recombination between homologous IS elements identified in a large collection of Escherichia coli mutation accumulation lines by analysis of whole genome shotgun sequencing data. Based on 857 identified events (758 IS insertions, 98 recombinations and 1 excision), we estimate that the rate of IS insertion is 3.5 × 10⁻⁴ insertions per genome per generation and the rate of IS homologous recombination is 4.5 × 10⁻⁵ recombinations per genome per generation. These events are mostly contributed by the IS elements IS1, IS2, IS5 and IS186. Spatial analysis of new insertions suggest that transposition is biased to proximal insertions, and the length spectrum of IS-caused deletions is largely explained by local hopping. For any of the ISs studied there is no region of the circular genome that is favored or disfavored for new insertions but there are notable hotspots for deletions. Some elements have preferences for non-coding sequence or for the beginning and end of coding regions, largely explained by target site motifs. Interestingly, transposition and deletion rates remain constant across the wild-type and 12 mutant E. coli lines, each deficient in a distinct DNA repair pathway. Finally, we characterized the target sites of four IS families, confirming previous results and characterizing a highly specific pattern at IS186 target-sites, 5′-GGGG(N6/N7)CCCC-3′. We also detected 48 long deletions not involving IS elements.     

Estimation of the Spontaneous Mutation Rate per Nucleotide Site in a Drosophila melanogaster Full-Sib Family

We employed deep genome sequencing of two parents and 12 of their offspring to estimate the mutation rate per site per generation in a full-sib family of Drosophila melanogaster recently sampled from a natural population. Sites that were homozygous for the same allele in the parents and heterozygous in one or more offspring were categorized as candidate mutations and subjected to detailed analysis. In 1.23 × 10⁹ callable sites from 12 individuals, we confirmed six single nucleotide mutations. We estimated the false negative rate in the experiment by generating synthetic mutations using the empirical distributions of numbers of nonreference bases at heterozygous sites in the offspring. The proportion of synthetic mutations at callable sites that we failed to detect was <1%, implying that the false negative rate was extremely low. Our estimate of the point mutation rate is 2.8 × 10⁻⁹ (95% confidence interval = 1.0 × 10⁻⁹ − 6.1 × 10⁻⁹) per site per generation, which is at the low end of the range of previous estimates, and suggests an effective population size for the species of ∼1.4 × 10⁶. At one site, point mutations were present in two individuals, indicating that there had been a premeiotic mutation cluster, although surprisingly one individual had a G→A transition and the other a G→T transversion, possibly associated with error-prone mismatch repair. We also detected three short deletion mutations and no insertions, giving a deletion mutation rate of 1.2 × 10⁻⁹ (95% confidence interval = 0.7 × 10⁻⁹ − 11 × 10⁻⁹).     

An Age-of-Allele Test of Neutrality for Transposable Element Insertions

How natural selection acts to limit the proliferation of transposable elements (TEs) in genomes has been of interest to evolutionary biologists for many years. To describe TE dynamics in populations, previous studies have used models of transposition–selection equilibrium that assume a constant rate of transposition. However, since TE invasions are known to happen in bursts through time, this assumption may not be reasonable. Here we propose a test of neutrality for TE insertions that does not rely on the assumption of a constant transposition rate. We consider the case of TE insertions that have been ascertained from a single haploid reference genome sequence. By conditioning on the age of an individual TE insertion allele (inferred by the number of unique substitutions that have occurred within the particular TE sequence since insertion), we determine the probability distribution of the insertion allele frequency in a population sample under neutrality. Taking models of varying population size into account, we then evaluate predictions of our model against allele frequency data from 190 retrotransposon insertions sampled from North American and African populations of Drosophila melanogaster. Using this nonequilibrium neutral model, we are able to explain ∼80% of the variance in TE insertion allele frequencies based on age alone. Controlling for both nonequilibrium dynamics of transposition and host demography, we provide evidence for negative selection acting against most TEs as well as for positive selection acting on a small subset of TEs. Our work establishes a new framework for the analysis of the evolutionary forces governing large insertion mutations like TEs, gene duplications, or other copy number variants.     

Repeatability of adaptation in experimental populations of different sizes

The degree to which evolutionary trajectories and outcomes are repeatable across independent populations depends on the relative contribution of selection, chance and history. Population size has been shown theoretically and empirically to affect the amount of variation that arises among independent populations adapting to the same environment. Here, we measure the contribution of selection, chance and history in different-sized experimental populations of the unicellular alga Chlamydomonas reinhardtii adapting to a high salt environment to determine which component of evolution is affected by population size. We find that adaptation to salt is repeatable at the fitness level in medium (N_e = 5 × 10⁴) and large (N_e = 4 × 10⁵) populations because of the large contribution of selection. Adaptation is not repeatable in small (N_e = 5 × 10³) populations because of large constraints from history. The threshold between stochastic and deterministic evolution in this case is therefore between effective population sizes of 10³ and 10⁴. Our results indicate that diversity across populations is more likely to be maintained if they are small. Experimental outcomes in large populations are likely to be robust and can inform our predictions about outcomes in similar situations.     

Ideotype Population Exploration: Growth,Photosynthesis, and Yield Components at Different Planting Densities in Winter Oilseed Rape (Brassica napus L.)

Rapeseed is one of the most important edible oil crops in the world and the seed yield has lagged behind the increasing demand driven by population growth. Winter oilseed rape (Brassica napus L.) is widely cultivated with relatively low yield in China, so it is necessary to find the strategies to improve the expression of yield potential. Planting density has great effects on seed yield of crops. Hence, field experiments were conducted in Wuhan in the Yangtze River basin with one conventional variety (Zhongshuang 11, ZS11) and one hybrid variety (Huayouza 9, HYZ9) at five planting densities (27.0×10⁴, 37.5×10⁴, 48.0×10⁴, 58.5×10⁴, 69.0×10⁴ plants ha^–1) during 2010–2012 to investigate the yield components. The physiological traits for high-yield and normal-yield populations were measured during 2011–2013. Our results indicated that planting densities of 58.5×10⁴ plants ha^–1 in ZS11 and 48.0×10⁴ plants ha^–1 in HYZ9 have significantly higher yield compared with the density of 27.0×10⁴ plants ha^–1for both varieties. The ideal silique numbers for ZS11 and HYZ9 were ∼0.9×10⁴ (n m^–2) and ∼1×10⁴ (n m^-2), respectively, and ideal primary branches for ZS11 and HYZ9 were ∼250 (n m^–2) and ∼300 (n m^–2), respectively. The highest leaf area index (LAI) and silique wall area index (SAI) was ∼5.0 and 7.0, respectively. Moreover, higher leaf net photosynthetic rate (Pn) and water use efficiency (WUE) were observed in the high-yield populations. A significantly higher level of silique wall photosynthesis and rapid dry matter accumulation were supposed to result in the maximum seed yield. Our results suggest that increasing the planting density within certain range is a feasible approach for higher seed yield in winter rapeseed in China.     

Genome-wide development of transposable elements-based markers in foxtail millet and construction of an integrated database

Transposable elements (TEs) are major components of plant genome and are reported to play significant roles in functional genome diversity and phenotypic variations. Several TEs are highly polymorphic for insert location in the genome and this facilitates development of TE-based markers for various genotyping purposes. Considering this, a genome-wide analysis was performed in the model plant foxtail millet. A total of 30,706 TEs were identified and classified as DNA transposons (24,386), full-length Copia type (1,038), partial or solo Copia type (10,118), full-length Gypsy type (1,570), partial or solo Gypsy type (23,293) and Long- and Short-Interspersed Nuclear Elements (3,659 and 53, respectively). Further, 20,278 TE-based markers were developed, namely Retrotransposon-Based Insertion Polymorphisms (4,801, ∼24%), Inter-Retrotransposon Amplified Polymorphisms (3,239, ∼16%), Repeat Junction Markers (4,451, ∼22%), Repeat Junction-Junction Markers (329, ∼2%), Insertion-Site-Based Polymorphisms (7,401, ∼36%) and Retrotransposon-Microsatellite Amplified Polymorphisms (57, 0.2%). A total of 134 Repeat Junction Markers were screened in 96 accessions of Setaria italica and 3 wild Setaria accessions of which 30 showed polymorphism. Moreover, an open access database for these developed resources was constructed (Foxtail millet Transposable Elements-based Marker Database; http://59.163.192.83/ltrdb/index.html). Taken together, this study would serve as a valuable resource for large-scale genotyping applications in foxtail millet and related grass species.     

Low-temperature leaf photosynthesis of a Miscanthus germplasm collection correlates positively to shoot growth rate and specific leaf area

Background and Aims The C₄ perennial grass miscanthus has been found to be less sensitive to cold than most other C₄ species, but still emerges later in spring than C₃ species. Genotypic differences in miscanthus were investigated to identify genotypes with a high cold tolerance at low temperatures and quick recovery upon rising temperatures to enable them to exploit the early growing season in maritime cold climates. Suitable methods for field screening of cold tolerance in miscanthus were also identified.Methods Fourteen genotypes of M. sacchariflorus, M. sinensis, M. tinctorius and M. × giganteus were selected and grown under warm (24 °C) and cold (14 °C) conditions in a controlled environment. Dark-adapted chlorophyll fluorescence, specific leaf area (SLA) and net photosynthetic rate at a photosynthetically active radiation (PAR) of 1000 μmol m^–2 s^–1 (A₁₀₀₀) were measured. Photosynthetic light and CO₂ response curves were obtained from 11 of the genotypes, and shoot growth rate was measured under field conditions.Key Results A positive linear relationship was found between SLA and light-saturated photosynthesis (A_sat) across genotypes, and also between shoot growth rate under cool field conditions and A₁₀₀₀ at 14 °C in a climate chamber. When lowering the temperature from 24 to 14 °C, one M. sacchariflorus exhibited significantly higher A_sat and maximum photosynthetic rate in the CO₂ response curve (V_max) than other genotypes at 14 °C, except M. × giganteus ‘Hornum’. Several genotypes returned to their pre-chilling A₁₀₀₀ values when the temperature was increased to 24 °C after 24 d growth at 14 °C.Conclusions One M. sacchariflorus genotype had similar or higher photosynthetic capacity than M. × giganteus, and may be used for cultivation together with M. × giganteus or for breeding new interspecies hybrids with improved traits for temperate climates. Two easily measured variables, SLA and shoot growth rate, may be useful for genotype screening of productivity and cold tolerance.     

Whole Genome Sequencing of Mutation Accumulation Lines Reveals a Low Mutation Rate in the Social Amoeba Dictyostelium discoideum

Spontaneous mutations play a central role in evolution. Despite their importance, mutation rates are some of the most elusive parameters to measure in evolutionary biology. The combination of mutation accumulation (MA) experiments and whole-genome sequencing now makes it possible to estimate mutation rates by directly observing new mutations at the molecular level across the whole genome. We performed an MA experiment with the social amoeba Dictyostelium discoideum and sequenced the genomes of three randomly chosen lines using high-throughput sequencing to estimate the spontaneous mutation rate in this model organism. The mitochondrial mutation rate of 6.76×10⁻⁹, with a Poisson confidence interval of 4.1×10⁻⁹ − 9.5×10⁻⁹, per nucleotide per generation is slightly lower than estimates for other taxa. The mutation rate estimate for the nuclear DNA of 2.9×10⁻¹¹, with a Poisson confidence interval ranging from 7.4×10⁻¹³ to 1.6×10⁻¹⁰, is the lowest reported for any eukaryote. These results are consistent with low microsatellite mutation rates previously observed in D. discoideum and low levels of genetic variation observed in wild D. discoideum populations. In addition, D. discoideum has been shown to be quite resistant to DNA damage, which suggests an efficient DNA-repair mechanism that could be an adaptation to life in soil and frequent exposure to intracellular and extracellular mutagenic compounds. The social aspect of the life cycle of D. discoideum and a large portion of the genome under relaxed selection during vegetative growth could also select for a low mutation rate. This hypothesis is supported by a significantly lower mutation rate per cell division in multicellular eukaryotes compared with unicellular eukaryotes.     

Pathogenicity of Five Strains of Toxoplasma gondii from Different Animals to Chickens

Toxoplasma gondii is a protozoan parasite with a broad range of intermediate hosts. Chickens as important food-producing animals can also serve as intermediate hosts. To date, experimental studies on the pathogenicity of T. gondii in broiler chickens were rarely reported. The objective of the present study was to compare the pathogenicity of 5 different T. gondii strains (RH, CN, JS, CAT2, and CAT3) from various host species origin in 10-day-old chickens. Each group of chickens was infected intraperitoneally with 5×10⁸, 1×10⁸, 1×10⁷, and 1×10⁶ tachyzoites of the 5 strains, respectively. The negative control group was mockly inoculated with PBS alone. After infection, clinical symptoms and rectal temperatures of all the chickens were checked daily. Dead chickens during acute phage of the infection were checked for T. gondii tachyzoites by microscope, while living cases were checked for T. gondii infection at day 53 post-inoculation (PI) by PCR method. Histopathological sections were used to observe the pathological changes in the dead chickens and the living animals at day 53 PI. No significant differences were found in survival periods, histopathological findings, and clinical symptoms among the chickens infected with the RH, CN, CAT2, and CAT3 strains. Histopathological findings and clinical symptoms of the JS (chicken origin) group were similar to the others. However, average survival times of infected chickens of the JS group inoculated with 5×10⁸ and 1×10⁸ tachyzoites were 30.0 and 188.4 hr, respectively, significantly shorter than those of the other 4 mammalian isolates. Chickens exposed to 10⁸ of T. gondii tachyzoites and higher showed acute signs of toxoplasmosis, and the lesions were relatively more severe than those exposed to lower doses. The results indicated that the pathogenicity of JS strain was comparatively stronger to the chicken, and the pathogenicity was dose-dependent.     

Sensitivity of Plasmodium falciparum to Antimalarial Drugs in Hainan Island,China

Pyronaridine and artesunate have been shown to be effective in falciparum malaria treatment. However, pyronaridine is rarely used in Hainan Island clinically, and artesunate is not widely used as a therapeutic agent. Instead, conventional antimalarial drugs, chloroquine and piperaquine, are used, explaining the emergence of chloroquine-resistant Plasmodium falciparum. In this article, we investigated the sensitivity of P. falciparum to antimalarial drugs used in Hainan Island for rational drug therapy. We performed in vivo (28 days) and in vitro tests to determine the sensitivity of P. falciparum to antimalarial drugs. Total 46 patients with falciparum malaria were treated with dihydroartemisinin/piperaquine phosphate (DUO-COTECXIN) and followed up for 28 day. The cure rate was 97.8%. The mean fever clearance time (22.5±10.6 hr) and the mean parasite clearance time (27.3±12.2 hr) showed no statistical significance with different genders, ages, temperatures, or parasite density (P>0.05). The resistance rates of chloroquine, piperaquine, pyronarididine, and artesunate detected in vitro were 71.9%, 40.6%, 12.5%, and 0%, respectively (P<0.0001). The resistance intensities decreased as follows: chloroquine>piperaquine>pyronarididine>artesunate. The inhibitory dose 50 (IC₅₀) was 3.77×10^-6 mol/L, 2.09×10^-6 mol/L, 0.09×10^-6 mol/L, and 0.05×10^-6 mol/L, and the mean concentrations for complete inhibition (CIMC) of schizont formation were 5.60×10^-6 mol/L, 9.26×10^-6 mol/L, 0.55×10^-6 mol/L, and 0.07×10^-6 mol/L, respectively. Dihydroartemisinin showed a strong therapeutic effect against falciparum malaria with a low toxicity.     

Implications of fungal infections and mycotoxins in camel diseases in Saudi Arabia

Natural feed ingredients (corn, barley and wheat bran) and compound feed (manufactured pellet) are two types of fodder used for animal feeding, especially camel in Saudi Arabia. Twenty samples of each type of fodder were collected from seven different regions and screened for the presence of fungi, aflatoxins, ochratoxin and zearalenone. Fungal isolation of natural feed ingredients yielded 10 genera and 38 species of different fungi. Compound fodder samples were contaminated with 16 genera and 32 species of fungi. Total counts of Aspergillus, Penicillium and Fusarium in the animal feed samples were ranged from 54 to 223 × 10³, 31.9 to 60 × 10³ and 18 to 29 × 10³ CFU/g, respectively. These isolates when tested for aflatoxin, ochratoxin and zearalenone producing ability, revealed this property in only four isolate, identified as Aspergillus flavus, A. parasiticus, A. ochraceus and Fusarium graminaerum. The percentage of toxigenic fungi was ranged from 5.5% to 30% for natural feed ingredients and from 4.5% to 20% for compound feed. The incidence of aflatoxins (AFT) in samples of natural feed ingredients was found to be ranged from 1 to 24.8 ppb, ochratoxin A (OTA) ranged from 1 to 44 ppb and zearalenone (ZON) ranged from 1 to 23 ppb. Contamination of compound feed with aflatoxin and ochratoxin A was ranged from 1 to 6.4 ppb and 1 to 4.7 ppb, respectively. All samples collected were found contaminated with fungi or their toxins and natural feed samples were more contaminated compared to compound feed samples. The concentrations detected were in the allowed limit (<20 ppb) except four samples of natural feed ingredients which were above the allowed limit of the tested mycotoxins. In conclusion, feed samples were contaminated with fungi and some toxigenic isolates which were responsible about mycotoxin production. Some samples had exceeded amount of AFT, OTA and ZON and may be contaminated with other mycotoxins which mean implication of fungi in camel health problems and death in Saudi Arabia.     

High-Temperature-Induced Transposition of Insertion Elements in Burkholderia multivorans ATCC 17616

An efficient and quantitative method to analyze the transposition of various insertion sequence (IS) elements in Burkholderia multivorans ATCC 17616 was devised. pGEN500, a plasmid carrying a Bacillus subtilis-derived sacB gene, was introduced into ATCC 17616 cells, and 25% of their sucrose-resistant derivatives were found to carry various IS elements on pGEN500. A PCR-based experimental protocol, in which a mixture of several specific primer pairs was used, revealed that pGEN500 captured, in addition to five previously reported IS elements (IS401, IS402, IS406, IS407, and IS408), three novel IS elements, ISBmu1, ISBmu2, and ISBmu3. The global transposition frequency of these IS elements was enhanced more than sevenfold under a high-temperature condition (42°C) but not under oxidative stress or starvation conditions. To our knowledge, this is the first report demonstrating the elevated transposition activities of several IS elements at a high temperature. The efficient experimental protocol developed in this study will be useful in quantitatively and simultaneously investigating various IS elements, as well as in capturing novel functional mobile elements from a wide variety of bacteria.     

Nitric-oxide Dioxygenase Function of Human Cytoglobin with Cellular Reductants and in Rat Hepatocytes

Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b₅ were found to support a high NOD activity. Cygb-NOD activity shows respective K_m values for ascorbate, cytochrome b₅, NO, and O₂ of 0.25 mm, 0.3 μm, 40 nm, and ∼20 μm and achieves a k_cat of 0.5 s⁻¹. Ascorbate and cytochrome b₅ reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m⁻¹ s⁻¹ and 3 × 10⁶ m⁻¹ s⁻¹, respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar k_cat of 1.2 s⁻¹, a K_m(NO) of 40 nm, and a k_cat/K_m(NO) (k′_NOD) value of 3 × 10⁷ m⁻¹ s⁻¹, demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O₂] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b₅ as reductants.     

Effects of heat and UV radiation on the mobilization of transposon mariner-Mos1

There are many complex interactions between transposable elements (TEs) and host genomes. Environmental changes that induce stressful conditions help to contribute for increasing complexity of these interactions. The transposon mariner-Mos1 increases its mobilization under mild heat stress. It has putative heat shock elements (HSEs), which are probably activated by heat shock factors (HSFs). Ultraviolet radiation (UVC) is a stressor that has been suggested as able to activate heat shock protein genes (Hsp). In this study, we test the hypothesis that if UVC induces Hsp expression, as heat does, it could also promote mariner-Mos1 transposition and mobilization. The Drosophila simulans white-peach is a mutant lineage that indicates the mariner-Mos1 transposition phenotypically through the formation of mosaic eyes. This lineage was exposed to UVC or mild heat stress (28 °C) in order to evaluate the induction of mariner-Mos1 expression by RT-qPCR, as well as the mariner-Mos1 mobilization activity based on the count number of red spots in the eyes. The effects of both treatments on the developmental time of flies and cell cycle progression were also investigated. Both the analysis of eyes and mariner-Mos1 gene expression indicate that UVC radiation has no effect in mariner-Mos1 transposition, although heat increases the expression and mobilization of this TE soon after the treatment. However, the expression of Hsp70 gene increased after 24 h of UVC exposure, suggesting different pathway of activation. These results showed that heat promotes mariner-Mos1 mobilization, although UVC does not induce the expression or mobilization of this TE.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0611-2) contains supplementary material, which is available to authorized users.     

Low levels of realized seed and pollen gene flow and strong spatial genetic structure in a small,isolated and fragmented population of the tropical tree Copaifera langsdorffii Desf

Over the past century, the Brazilian Atlantic forest has been reduced to small, isolated fragments of forest. Reproductive isolation theories predict a loss of genetic diversity and increases in inbreeding and spatial genetic structure (SGS) in such populations. We analysed eight microsatellite loci to investigate the pollen and seed dispersal patterns, genetic diversity, inbreeding and SGS of the tropical tree Copaifera langsdorffii in a small (4.8 ha), isolated population. All 112 adult trees and 128 seedlings found in the stand were sampled, mapped and genotyped. Seedlings had significantly lower levels of genetic diversity (A=16.5±0.45, mean±95% s.e.; H_e=0.838±0.006) than did adult trees (A=23.2±0.81; H_e=0.893±0.030). Parentage analysis did not indicate any seed immigration (m_seeds=0) and the pollen immigration rate was very low (m_pollen=0.047). The average distance of realized pollen dispersal within the stand was 94 m, with 81% of the pollen travelling <150 m. A significant negative correlation was found between the frequency and distance of pollen dispersal (r=−0.79, P<0.01), indicating that short-distance pollinations were more frequent. A significant SGS for both adults (∼50 m) and seedlings (∼20 m) was also found, indicating that most of the seeds were dispersed over short distances. The results suggested that the spatial isolation of populations by habitat fragmentation can restrict seed and pollen gene flow, increase SGS and affect the genetic diversity of future generations.     

Integration of New Genes into Cellular Networks,and Their Structural Maturation

It has been recently discovered that new genes can originate de novo from noncoding DNA, and several biological traits including expression or sequence composition form a continuum from noncoding sequences to conserved genes. In this article, using yeast genes I test whether the integration of new genes into cellular networks and their structural maturation shows such a continuum by analyzing their changes with gene age. I show that 1) The number of regulatory, protein–protein, and genetic interactions increases continuously with gene age, although with very different rates. New regulatory interactions emerge rapidly within a few million years, while the number of protein–protein and genetic interactions increases slowly, with a rate of 2–2.25 × 10⁻⁸/year and 4.8 × 10⁻⁸/year, respectively. 2) Gene essentiality evolves relatively quickly: the youngest essential genes appear in proto-genes ∼14 MY old. 3) In contrast to interactions, the secondary structure of proteins and their robustness to mutations indicate that new genes face a bottleneck in their evolution: proto-genes are characterized by high β-strand content, high aggregation propensity, and low robustness against mutations, while conserved genes are characterized by lower strand content and higher stability, most likely due to the higher probability of gene loss among young genes and accumulation of neutral mutations.     

A new species of plasmodiidae (Coccidia: Hemosporidia) from the blood of the skink Scincus hemprichii (Scincidae: Reptilia) in Saudi Arabia

Fallisia arabica n. sp. was described from peripheral blood smears of the Skink lizard, Scincus hemprichii from Jazan Province in the southwest of Saudi Arabia. Schizogony and gametogony take place within neutrophils in the peripheral blood of the host. Mature schizont is rosette shaped 17.5 ± 4.1 × 17.0 ± 3.9 μm, with a L/W ratio of 1.03(1.02–1.05) μm and produces 24(18–26) merozoites. Young gametocytes are ellipsoidal, 5.5 ± 0.8 × 3.6 ± 0.5 μm, with a L/W of 1.53(1.44–1.61) μm. Mature macrogametocytes are ellipsoidal, 9.7 ± 1.2 × 7.8 ± 1.0 μm, with a L/W of 1.24(1.21–1.34) μm and microgametocytes are ellipsoidal, 7.0 ± 1.1 × 6.8 ± 0.9 μm. with a L/W of 1.03(1.01–1.10) μm. In comparison to the described Fallisia species, this new taxon has rosette schizonts and is larger than F. dominicensis, in Hispaniola, F. bipocrati, F. poecilopi, in Panama, F. thecadactyli in Venezuela, and F. effusa, F. simplex, F. modesta, in Brazil. F. arabica has fewer merozoites than F. effusa, F. poecilopi, F. thecadactyli and F. siamense in Thailand. This new species has more merozoites than F. dominicensis and F. modesta. All of these species belong to diverse saurian families (Agamidae, Gekkonidae, Polychrotidae, Scincidae and Teiidae) parasitize only thrombocytes or lymphocytes and some species parasitize immature erythroid cells and leucocytes.     

Multiplex-Touchdown PCR to Simultaneously Detect Cryptosporidium parvum,Giardia lamblia,and Cyclospora cayetanensis,the Major Causes of Traveler’s Diarrhea

This study aimed to develop a multiplex-touchdown PCR method to simultaneously detect 3 species of protozoan parasites, i.e., Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the major causes of traveler’s diarrhea and are resistant to standard antimicrobial treatments. The target genes included the Cryptosporidium oocyst wall protein for C. parvum, Glutamate dehydrogenase for G. lamblia, and 18S ribosomal RNA (18S rRNA) for C. cayetanensis. The sizes of the amplified fragments were 555, 188, and 400 bps, respectively. The multiplex-touchdown PCR protocol using a primer mixture simultaneously detected protozoa in human stools, and the amplified gene was detected in >1×10³ oocysts for C. parvum, >1×10⁴ cysts for G. lamblia, and >1 copy of the 18S rRNA gene for C. cayetanensis. Taken together, our protocol convincingly demonstrated the ability to simultaneously detect C. parvum, G. lamblia, and C. cayetanenesis in stool samples.     

Genetic Architecture of Skin and Eye Color in an African-European Admixed Population

Variation in human skin and eye color is substantial and especially apparent in admixed populations, yet the underlying genetic architecture is poorly understood because most genome-wide studies are based on individuals of European ancestry. We study pigmentary variation in 699 individuals from Cape Verde, where extensive West African/European admixture has given rise to a broad range in trait values and genomic ancestry proportions. We develop and apply a new approach for measuring eye color, and identify two major loci (HERC2[OCA2] P = 2.3×10⁻⁶², SLC24A5 P = 9.6×10⁻⁹) that account for both blue versus brown eye color and varying intensities of brown eye color. We identify four major loci (SLC24A5 P = 5.4×10⁻²⁷, TYR P = 1.1×10⁻⁹, APBA2[OCA2] P = 1.5×10⁻⁸, SLC45A2 P = 6×10⁻⁹) for skin color that together account for 35% of the total variance, but the genetic component with the largest effect (∼44%) is average genomic ancestry. Our results suggest that adjacent cis-acting regulatory loci for OCA2 explain the relationship between skin and eye color, and point to an underlying genetic architecture in which several genes of moderate effect act together with many genes of small effect to explain ∼70% of the estimated heritability.     

Mobility and Generation of Mosaic Non-Autonomous Transposons by Tn3-Derived Inverted-Repeat Miniature Elements (TIMEs)

Functional transposable elements (TEs) of several Pseudomonas spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i) 13 insertion sequences from 5 IS families (IS3, IS5, ISL3, IS30 and IS1380), (ii) isoforms of two Tn3-family transposons – Tn5563a and Tn4662a (the latter contains a toxin-antitoxin system), as well as (iii) non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs), highly conserved in Tn5563a and many other Tn3-family transposons. The simplest elements of this type, designated TIMEs (Tn3 family-derived Inverted-repeat Miniature Elements) (262 bp), were identified within two natural plasmids (pZM1P1 and pLM8P2) of Pseudomonas spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element “captured” with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a res site typical for many Tn3-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and evolution, not only of transposons and plasmids, but also of other types of mobile genetic elements.