Subtelomeric plasticity contributes to gene family expansion in the human parasitic flatworm Schistosoma mansoni.

BACKGROUND: The genomic region that lies between the telomere and chromosome body, termed the subtelomere, is heterochromatic, repeat-rich, and frequently undergoes rearrangement. Within this region, large-scale structural changes enable gene diversification, and, as such, large multicopy gene families are often found at the subtelomere. In some parasites, genes associated with proliferation, invasion, and survival are often found in these regions, where they benefit from the subtelomere's highly plastic, rapidly changing nature. The increasing availability of complete (or near complete) parasite genomes provides an opportunity to investigate these typically poorly defined and overlooked genomic regions and potentially reveal relevant gene families necessary for the parasite's lifestyle. RESULTS: Using the latest chromosome-scale genome assembly and hallmark repeat richness observed at chromosome termini, we have identified and characterised the subtelomeres of Schistosoma mansoni, a metazoan parasitic flatworm that infects over 250 million people worldwide. Approximately 12% of the S. mansoni genome is classified as subtelomeric, and, in line with other organisms, we find these regions to be gene-poor but rich in transposable elements. We find that S. mansoni subtelomeres have undergone extensive interchromosomal recombination and that these sites disproportionately contribute to the 2.3% of the genome derived from segmental duplications. This recombination has led to the expansion of subtelomeric gene clusters containing 103 genes, including the immunomodulatory annexins and other gene families with unknown roles. The largest of these is a 49-copy plexin domain-containing protein cluster, exclusively expressed in the tegument-the tissue located at the host-parasite physical interface-of intramolluscan life stages. CONCLUSIONS: We propose that subtelomeric regions act as a genomic playground for trial-and-error of gene duplication and subsequent divergence. Owing to the importance of subtelomeric genes in other parasites, gene families implicated in this subtelomeric expansion within S. mansoni warrant further characterisation for a potential role in parasitism.

Phenotypic and genotypic analysis of benzimidazole resistance in reciprocal genetic crosses of Haemonchus contortus.

Haemonchus contortus is arguably one of the most economically important and ubiquitous parasites of livestock globally and commonly involved in cases of anthelmintic resistance. Here, we performed reciprocal genetic crosses using susceptible (MHco3(ISE)) and multiple anthelmintic resistant (MHco18(UGA2004)) H. contortus isolates. Resultant admixed populations were designated MHco3/18 or MHco18/3, where the lead isolate reflects the origin of the females. Three independent filial generations were generated for each cross, which were subjected to bioassays, molecular approaches and population genetic analyses to investigate the phenotypic and genotypic inheritance of benzimidazole (BZ) resistance at each stage. A panel of microsatellite markers confirmed the success of the genetic cross as markers from both parents were seen in the F1 crosses. Egg hatch tests revealed a stark difference between the two F1 crosses with ED50 estimates for MHco18/3 being 9 times greater than those for MHco3/18. Resistance factors based on ED50 estimates ranged from 6 to 57 fold in the filial progeny compared to MHco3(ISE) parents. Molecular analysis of the F167Y and F200Y SNP markers associated with BZ resistance were analysed by pyrosequencing and MiSeq deep amplicon sequencing, which showed that MHco3/18.F1 and MHco18/3.F1 both had similar frequencies of the F200Y resistant allele (45.3% and 44.3%, respectively), whereas for F167Y, MHco18/3.F1 had a two-fold greater frequency of the resistant-allele compared to MHco3/18.F1 (18.2% and 8.8%, respectively). Comparison between pyrosequencing and MiSeq amplicon sequencing revealed that the allele frequencies derived from both methods were concordant at codon 200 (rc = 0.97), but were less comparable for codon 167 (rc = 0.55). The use of controlled reciprocal genetic crosses have revealed a potential difference in BZ resistance phenotype dependent on whether the resistant allele is paternally or maternally inherited. These findings provide new insight and prompt further investigation into the inheritance of BZ resistance in H. contortus.

The global diversity of Haemonchus contortus is shaped by human intervention and climate.

Haemonchus contortus is a haematophagous parasitic nematode of veterinary interest. We have performed a survey of its genome-wide diversity using single-worm whole genome sequencing of 223 individuals sampled from 19 isolates spanning five continents. We find an African origin for the species, together with evidence for parasites spreading during the transatlantic slave trade and colonisation of Australia. Strong selective sweeps surrounding the ß-tubulin locus, a target of benzimidazole anthelmintic drug, are identified in independent populations. These sweeps are further supported by signals of diversifying selection enriched in genes involved in response to drugs and other anthelmintic-associated biological functions. We also identify some candidate genes that may play a role in ivermectin resistance. Finally, genetic signatures of climate-driven adaptation are described, revealing a gene acting as an epigenetic regulator and components of the dauer pathway. These results begin to define genetic adaptation to climate in a parasitic nematode.

Effects of glyphosate formulations on the population dynamics of two freshwater cladoceran species.

The general objective of this work is to experimentally assess the effects of acute glyphosate pollution on two freshwater cladoceran species (Daphnia magna and Ceriodaphnia dubia) and to use this information to predict the population dynamics and the potential for recovery of exposed organisms. Five to six concentrations of four formulations of glyphosate (4-Gly) (Eskoba®, Panzer Gold®, Roundup Ultramax® and Sulfosato Touchdown®) were evaluated in both cladoceran species through acute tests and 15-day recovery tests in order to estimate the population dynamics of microcrustaceans. The endpoints of the recovery test were: survival, growth (number of molts), fecundity, and the intrinsic population growth rate (r). A matrix population model (MPM) was applied to r of the survivor individuals of the acute tests, followed by a Monte Carlo simulation study. Among the 4-Gly tested, Sulfosato Touchdown® was the one that showed higher toxicity, and C. dubia was the most sensitive species. The Monte Carlo simulation study showed an average value of λ always <1 for D. magna, indicating that its populations would not be able to survive under natural environmental conditions after an acute Gly exposure between 0.25 and 35 a.e. mg L-1. The average value of λ for C. dubia was also <1 after exposure to Roundup Ultramax®: 1.30 and 1.20 for 1.21 and 2.5 mg a.e. L-1,respectively. The combined methodology-recovery tests and the later analysis through MPM with a Monte Carlo simulation study-is proposed to integrate key demographic parameters and predict the possible fate of microcrustacean populations after being exposed to acute 4-Gly contamination events.

False-positive error rates in routine application of repeated measurements ANOVA.

Failure to recognize the serious implications of heterogeneous correlations and disregard of the multiple test problem in interpreting the results from repeated measurements ANOVA of any single primary outcome measure can produce false-positive error rates that are more than five times the alpha level that is reported. Alternative analyses that do not depend on the symmetry assumption, together with a Bonferroni correction of the multiple tests of significance that are routinely accomplished by the repeated measurements ANOVA, appropriately control the probability of statistical support for a false-positive claim. The magnitudes of error inflation and appropriate procedures for error control are examined in this article using simulated clinical trials data.

Implications of chance baseline differences in repeated measurement designs.

Datasets representing randomized, parallel-groups designs were analyzed by repeated measurements ANOVA and linear trend analysis with and without baseline values being covaried. ANOVA tests for the between-groups main effect, groups X times interaction, and differences in linear trends across time periods are shown to be seriously conservative or seriously nonconservative, depending on the direction and significance of chance baseline mean difference. Inclusion of baseline scores as a covariate in the repeated measurements ANOVA provides appropriate correction for the between-groups (average) effect across time, but the covariate provides no correction for the within-subject effects that are concerned with differences in the rates or patterns of change across time. If one desires to evaluate differences between patterns of treatment-induced change, tests of significance for differences in group means on composite trend scores with covariance correction for baseline are recommended. If covariance correction is not or cannot be employed, the potentially "favorable" or "unfavorable" influence of chance baseline differences on tests of significance needs to be explicitly recognized.

A comment on the importance of numerical evaluation of analytic solutions involving approximations.

An analytic solution proposed by Senn (1) for removing the effects of covariate imbalance in controlled clinical trials was subjected to Monte Carlo evaluation. For practical applications of his derivation, Senn proposed substitution of sample statistics for parameters of the bivariate normal model. Unfortunately, that substitution produces severe distortion in the size of tests of significance for treatment effects when covariate imbalance is present. Numerical verification of proposed substitutions into analytic models is recommended as a prudent approach.

Estimating sample sizes for repeated measurement designs.

Formulas for estimating sample sizes that are required to provide specified power for analysis of variance (ANOVA) tests of significance in a two-group repeated measurements design are presented and evaluated. Power and sample size requirements depend on the pattern of treatment effects and the pattern of correlations among the repeated measurements, as well as on parameters common to sample size estimation for cross-sectional comparisons of treatment effects in simple randomized designs. Simplifying assumptions permit generation of these numerous parameter estimates from predictions of the magnitude of the standardized "effect size" at end of trial and the single correlation between the baseline and endpoint measurements. Monte Carlo methods are used to verify the actual power of different tests of significance for treatment effects in repeated measurement designs using sample sizes estimated by the formulas. The sample size implications of different patterns of treatment effects, levels of correlation, and numbers of repeated measurements are evaluated.