Flow cytometric analysis and microsatellite genotyping reveal extensive DNA content variation in Trypanosoma cruzi populations and expose contrasts between natural and experimental hybrids

Trypanosoma cruzi exhibits remarkable genetic heterogeneity. This is evident at the nucleotide level but also structurally, in the form of karyotypic variation and DNA content differences between strains. Although natural populations of T. cruzi are predominantly clonal, hybrid lineages (TcIId and TcIIe) have been identified and hybridisation has been demonstrated in vitro, raising the possibility that genetic exchange may continue to shape the evolution of this pathogen. The mechanism of genetic exchange identified in the laboratory is unusual, apparently involving fusion of diploid parents followed by genome erosion. We investigated DNA content diversity in natural populations of T. cruzi in the context of its genetic subdivisions by using flow cytometric analysis and multilocus microsatellite genotyping to determine the relative DNA content and estimate the ploidy of 54 cloned isolates. The maximum difference observed was 47.5% between strain Tu18 cl2 (TcIIb) and strain C8 cl1 (TcI), which we estimated to be equivalent to 73 Mb of DNA. Large DNA content differences were identified within and between discrete typing units (DTUs). In particular, the mean DNA content of TcI strains was significantly less than that for TcII strains (P < 0.001). Comparisons of hybrid DTUs TcIId/IIe with corresponding parental DTUs TcIIb/IIc indicated that natural hybrids are predominantly diploid. We also measured the relative DNA content of six in vitro-generated TcI hybrid clones and their parents. In contrast to TcIId/IIe hybrid strains these experimental hybrids comprised populations of sub-tetraploid organisms with mean DNA contents 1.65–1.72 times higher than the parental organisms. The DNA contents of both parents and hybrids were shown to be relatively stable after passage through a mammalian host, heat shock or nutritional stress. The results are discussed in the context of hybridisation mechanisms in both natural and in vitro settings.     

Genetic correlations between reproduction of crossbred ewes and the growth and carcass performance of their progeny

Genetic correlations were estimated between first cross ewe reproduction traits and growth and carcass traits of their second cross lamb progeny. The 2460 crossbred ewes were progeny of 74 maternal breed sires and mainly Merino dams. The ewes had 3 joinings to terminal sire rams with 6824 joining records resulting in 9002 lambs born and 7176 lambs slaughtered. The ewe reproduction traits included: fertility, litter size, ewe rearing ability or lamb survival, number of lambs born (NLBj) and weaned (NLWj) per ewe joined, with traits reflecting ewe productivity being total litter weight weaned (TWWj) per ewe joined and the component trait average lamb weaning weight in the litter (AWW). The lamb growth traits included weight of the lambs at birth (BWT), weaning (WWT) and post weaning (PWWT) as well as growth rate pre and post weaning. The lamb carcass traits included hot carcass weight (HCWT), dressing yield (DRESS%), fat depth (FatGR, FatC), eye muscle depth (EMD) and area (EMA) and meat quality traits (colour and pH). The genetic correlations were estimated by bivariate mixed models using ASReml. The genetic correlations between the composite reproduction traits of the ewes and the post-weaning growth rate of their lambs were high (0.67 for NLBj and 0.65 for NLWj). There were moderate positive correlations between NLWj and WWT (0.36), PWWT (0.49) and pre weaning growth (0.36) and NLBj with PWWT (0.31). BWT was negatively correlated with litter size (−0.34) and positively with ewe rearing ability (0.38). Most of the other genetic correlations were smaller than their standard errors which generally ranged from 0.2 to 0.3. The genetic correlations for HCWT with all the ewe reproduction traits were positive and moderate (0.29–0.53) and high with the ewe productivity traits that included weight of lambs (TWWj 0.98 and AWW 0.96). The genetic correlations among the other traits were variable and had high standard errors, generally ranging from 0.1 to 0.5. However there were generally negative and unfavourable genetic correlations between the reproduction traits and DRESS% and meat colour L*, whereas those with carcass fat (FatC and FatGR) were generally negative and favourable.     

Temporal trends in the discovery of human viruses

On average, more than two new species of human virus are reported every year. We constructed the cumulative species discovery curve for human viruses going back to 1901. We fitted a statistical model to these data; the shape of the curve strongly suggests that the process of virus discovery is far from complete. We generated a 95% credible interval for the pool of as yet undiscovered virus species of 38-562. We extrapolated the curve and generated an estimate of 10-40 new species to be discovered by 2020. Although we cannot predict the level of health threat that these new viruses will present, we conclude that novel virus species must be anticipated in public health planning. More systematic virus discovery programmes, covering both humans and potential animal reservoirs of human viruses, should be considered.     

Manual 768 or 384 well microplate gel 'dry' electrophoresis for PCR checking and SNP genotyping

Electrophoresis continues to be a mainstay in molecular genetic laboratories for checking, sizing and separating both PCR products, nucleic acids derived from in vivo or in vitro sources and nucleic acid-protein complexes. Many genomic and genetic applications demand high throughput, such as the checking of amplification products from many loci, from many clones, from many cell lines or from many individuals at once. These applications include microarray resource development and expression analysis, genome mapping, library and DNA bank screening, mutagenesis experiments and single nucleotide polymorphism (SNP) genotyping. PCR hardware compatible with industry standard 96 and 384 well microplates is commonplace. We have previously described a simple system for submerged horizontal 96 and 192 well polyacrylamide or agarose microplate array diagonal gel electrophoresis (MADGE) which is microplate compatible and suitable for PCR checking, SNP typing (restriction fragment length polymorphism or amplification refractory mutation system), microsatellite sizing and identification of unknown mutations. By substantial redesign of format and operations, we have derived an efficient 'dry' gel system that enables direct 96 pin manual transfer from PCR or other reactions in microplates, into 768 or 384 well gels. Combined with direct electrode contact in clamshell electrophoresis boxes which plug directly to contacts in a powered stacking frame and using 5-10 min electrophoresis times, it would be possible (given a sufficient supply of PCRs for examination) for 1 million gel tracks to be run per day for a minimal hardware investment and at minimal reagent costs. Applications of this system for PCR checking and SNP genotyping are illustrated.     

Origins of Chagas disease: Didelphis species are natural hosts of Trypanosoma cruzi I and armadillos hosts of Trypanosoma cruzi II, including hybrids

Trypanosoma cruzi, the causative agent of Chagas disease, has at least two principal intraspecific subdivisions, T. cruzi I (TCI) and T. cruzi II (TCII), the latter containing up to five subgroups (a-e). Whilst it is known that TCI predominates from the Amazon basin northwards and TCII to the South, where the disease is considered to be clinically more severe, the precise clinical and evolutionary significance of these divisions remains enigmatic. Here, we present compelling evidence of an association between TCI and opossums (Didelphis), and TCII and armadillos, on the basis of key new findings from the Paraguayan Chaco region, together with a comprehensive analysis of historical data. We suggest that the distinct arboreal and terrestrial ecologies, respectively, of these mammal hosts provide a persuasive explanation for the extant T. cruzi intraspecific diversity in South America, and for separate origins of Chagas disease in northern South America and in the southern cone countries.     

The effects of tracheal coiling on the vocalizations of cranes (Aves; Gruidae)

Summary It is generally supposed that the elongated, often coiled tracheae of many species of birds are adaptations for the production of loud, penetrating calls. A corollary supposition is that the acoustic effects are produced by the resonant properties of the elongated tube, with the birds being analogized to a wind instrument. We have experimented with several species of cranes possessing different degrees of tracheal coiling. Regardless of the degree of coiling, all cranes can utter extremely loud calls using remarkably low driving pressures. Neither surgical modifications of the trachea nor changing the respiratory gases to helium-oxygen produced consistent changes of voice that could be unambiguously attributed to changes of tubal resonances. However, shortening the trachea markedly reduced vocal intensity, the degree of reduction being roughly proportional to the degree of shortening. Although some of that reduction may derive from an increased impedance mismatch at the external aperture of the tube, and some from a decreased radiation directly from the hard walls of the trachea, these explanations scarcely account for the dramatic effects we observed. We, therefore, hypothesize a more unusual mechanism: The tracheal coils that are embedded in the sternum serve a function analogous to the bridge of a stringed instrument, transmitting the vibrations of a tiny sound source to a large radiating surface, the sternum. The sternum then vibrates against the large internal air reservoir of the avian airsac system. As it has a complex shape, the sternum will have many resonances and will respond to many frequencies; as a solid oscillator, its resonances will not be greatly affected by low density gases. Hence, we suggest that cranes and other birds with enlarged windpipes are more properly analogized with a violin than a trombone.     

Blood pressure, electrolyte composition and renal studies of pike, Esox lucius, following administration of salmon calcitonin

Pike were adapted to both fresh water and brackish water (salinity 2.2‰) conditions prior to measurements of blood pressure, electrolyte composition and renal function both before and after administration of either 3.75 μg calcitonin kg⁻¹ body weight or acetate saline. This protocol was designed to test the ability of calcitonin to affect significantly the fluid and electrolyte status of fish exposed to varying salinity. Whilst in freshwater-adapted animals the plasma content of calcium and magnesium was significantly depressed following calcitonin injection, no corresponding effect was seen following 2.2‰ salinity brackish water-adaptation. Moreover, no significant changes were apparent in renal function of pike adapted to either medium. It is concluded from these experiments that calcitonin in pike may play some part in extra-renal control of plasma calcium and magnesium content in fresh water but that it is unable to affect significantly the measured plasma ion content of brackish water-adapted animals.     

Metabolism of 4-chloro-2-methylphenoxyacetate by a soil pseudomonad. Ring-fission, lactonizing and delactonizing enzymes

1. A cell-free system, prepared from Pseudomonas N.C.I.B. 9340 grown on 4-chloro-2-methylphenoxyacetate (MCPA) was shown to catalyse the reaction sequence: 5-chloro-3-methylcatechol --> cis-cis-gamma-chloro-alpha-methylmuconate --> gamma-carboxymethylene-alpha-methyl-Delta(alphabeta)-butenolide --> gamma-hydroxy-alpha-methylmuconate. 2. The activity of the three enzymes involved in these reactions was completely resolved and the lactonizing and delactonizing enzymes were separated. 3. This part of the metabolic pathway of 4-chloro-2-methylphenoxyacetate is thus confirmed for this bacterium. 4. The ring-fission oxygenase required Fe(2+) or Fe(3+) and reduced glutathione for activity; the lactonizing enzyme is stimulated by Mn(2+), Mg(2+), Co(2+) and Fe(2+); no cofactor requirement could be demonstrated for the delactonizing enzyme. 5. cis-cis-gamma-Chloro-alpha-methylmuconic acid was isolated and found to be somewhat unstable, readily lactonizing to gamma-carboxymethylene-alpha-methyl-Delta(alphabeta)-butenolide. 6. Enzymically the lactonization appears to be a single-step dehydrochlorinase reaction.