No evidence for schistosome parasite fitness trade-offs in the intermediate and definitive host.

BACKGROUND: The trematode parasite Schistosoma mansoni uses an aquatic snail intermediate and a vertebrate definitive host to complete its life cycle. We previously showed that a key transmission trait-the number of cercariae larvae shed from infected Biomphalaria spp. snails-varies significantly within and between different parasite populations and is genetically controlled by five loci. We investigated the hypothesis that the success of parasite genotypes showing high propagative fitness in the intermediate snail host may be offset by lower reproductive fitness in the definitive vertebrate host. METHODS: We investigated this trade-off hypothesis by selecting parasite progeny producing high or low number of larvae in the snail and then comparing fitness parameters and virulence in the rodent host. We infected inbred BALB/c mice using two Schistosoma mansoni parasite lines [high shedder (HS) and low shedder (LS) lines] isolated from F2 progeny generated by genetic crosses between SmLE (HS parent) and SmBRE (LS parent) parasites. We used the F3 progeny to infect two populations of inbred Biomphalaria glabrata snails. We then compared life history traits and virulence of these two selected parasite lines in the rodent host to understand pleiotropic effects of genes determining cercarial shedding in parasites infecting the definitive host. RESULTS: HS parasites shed high numbers of cercariae, which had a detrimental impact on snail physiology (measured by laccase-like activity and hemoglobin rate), regardless of the snail genetic background. In contrast, selected LS parasites shed fewer cercariae and had a lower impact on snail physiology. Similarly, HS worms have a higher reproductive fitness and produced more viable F3 miracidia larvae than LS parasites. This increase in transmission is correlated with an increase in virulence toward the rodent host, characterized by stronger hepato-splenomegaly and hepatic fibrosis. CONCLUSIONS: These experiments revealed that schistosome parasite propagative and reproductive fitness was positively correlated in intermediate and definitive host (positive pleiotropy). Therefore, we rejected our trade-off hypothesis. We also showed that our selected schistosome lines exhibited low and high shedding phenotype regardless of the intermediate snail host genetic background. ​.

Hybrid inferiority and genetic incompatibilities drive divergence of fungal pathogens infecting the same host.

Agro-ecosystems provide environments that are conducive for rapid evolution and dispersal of plant pathogens. Previous studies have demonstrated that hybridization of crop pathogens can give rise to new lineages with altered virulence profiles. Currently, little is known about either the genetics of fungal pathogen hybridization or the mechanisms that may prevent hybridization between related species. The fungus Pyrenophora teres is a global pathogen of barley. The pathogenic fungus P. teres exists as two distinct lineages P. teres f. teres and P. teres f. maculata (Ptt and Ptm, respectively), which both infect barley but produce very distinct lesions and rarely interbreed. Interestingly, Ptt and Ptm can, by experimental mating, produce viable progenies. Here, we addressed the underlying genetics of reproductive barriers of P. teres. We hypothesize that Ptt and Ptm diverged in the past, possibly by adapting to distinct hosts, and only more recently colonized the same host in agricultural fields. Using experimental mating and in planta phenotyping in barley cultivars susceptible to both P. teres forms, we demonstrate that hybrids produce mixed infection phenotypes but overall show inferior pathogenic fitness relative to the pure parents. Based on analyses of 104 hybrid genomes, we identify signatures of negative epistasis between parental alleles at distinct loci (Dobzhansky-Müller incompatibilities). Most DMI regions are not involved in virulence but certain genes are predicted or known to play a role in virulence. These results potentially suggest that divergent niche adaptation-albeit in the same host plant-contributes to speciation in P. teres.

Bulk segregant linkage mapping for rodent and human malaria parasites.

In 2005 Richard Carter's group surprised the malaria genetics community with an elegant approach to rapidly mapping the genetic basis of phenotypic traits in rodent malaria parasites. This approach, which he termed "linkage group selection", utilized bulk pools of progeny, rather than individual clones, and exploited simple selection schemes to identify genome regions underlying resistance to drug treatment (or other phenotypes). This work was the first application of "bulk segregant" methodologies for genetic mapping in microbes: this approach is now widely used in yeast, and across multiple recombining pathogens ranging from Aspergillus fungi to Schistosome parasites. Genetic crosses of human malaria parasites (for which Richard Carter was also a pioneer) can now be conducted in humanized mice, providing new opportunities for exploiting bulk segregant approaches for a wide variety of malaria parasite traits. We review the application of bulk segregant approaches to mapping malaria parasite traits and suggest additional developments that may further expand the utility of this powerful approach.

Strains and approaches for genetic crosses in the oleaginous yeast Lipomyces starkeyi.

The oleaginous yeast Lipomyces starkeyi is a powerful lipid producer with great industrial potential. Recent studies have reported the isolation of mutant L. starkeyi cells with higher lipid producing capacity. Although genetic engineering strategies have been applied to L. starkeyi, classical genetic approaches are lacking. The development of tools that facilitate genetic crosses in L. starkeyi would not only make it possible to build improved lipid-producing strains but also facilitate molecular biological analysis of this species. In this study, I report a set of strains and approaches useful for performing genetic crosses with L. starkeyi. The homothallic L. starkeyi reportedly forms an ascus containing two to 20 spores. These spores were resistant to glusulase and could be dissected using a micromanipulator, suggesting that random spore and tetrad (spore dissection) analysis can be adapted for L. starkeyi. Additionally, to isolate a pair of heterothallic strains useful for genetic crosses, the homothallic strain was exposed to UV irradiation, and 10 self-sterile strains were crossed with one another. One of these combinations, Ls75 and Ls100, sporulated stably. Moreover, to detect genetic recombination, I introduced a different drug resistance marker into each strain and crossed them. The resulting progeny exhibited Mendelian segregation of the resistance markers. Altogether, the work reported here provides a powerful resource for genetic analysis in L. starkeyi.

Genetic architecture of transmission stage production and virulence in schistosome parasites.

Both theory and experimental data from pathogens suggest that the production of transmission stages should be strongly associated with virulence, but the genetic bases of parasite transmission/virulence traits are poorly understood. The blood fluke Schistosoma mansoni shows extensive variation in numbers of cercariae larvae shed and in their virulence to infected snail hosts, consistent with expected trade-offs between parasite transmission and virulence. We crossed schistosomes from two populations that differ 8-fold in cercarial shedding and in their virulence to Biomphalaria glabrata snail hosts, and determined four-week cercarial shedding profiles in F0 parents, F1 parents and 376 F2 progeny from two independent crosses in inbred snails. Sequencing and linkage analysis revealed that cercarial production is polygenic and controlled by five QTLs (i.e. Quantitative Trait Loci). These QTLs act additively, explaining 28.56% of the phenotypic variation. These results demonstrate that the genetic architecture of key traits relevant to schistosome ecology can be dissected using classical linkage mapping approaches.

Genomic and Genetic Approaches to Studying Antimalarial Drug Resistance and Plasmodium Biology.

Recent progress in genomics and molecular genetics has empowered novel approaches to study gene functions in disease-causing pathogens. In the human malaria parasite Plasmodium falciparum, the application of genome-based analyses, site-directed genome editing, and genetic systems that allow for temporal and quantitative regulation of gene and protein expression have been invaluable in defining the genetic basis of antimalarial resistance and elucidating candidate targets to accelerate drug discovery efforts. Using examples from recent studies, we review applications of some of these approaches in advancing our understanding of Plasmodium biology and illustrate their contributions and limitations in characterizing parasite genomic loci associated with antimalarial drug responses.

Handling Arabidopsis and Other Brassicaceae: Growth, Preservation of Seeds, Transformation, and Genetic Crosses.

Achieving optimal plant growth is essential for the advancement of Arabidopsis thaliana (Arabidopsis) research. Over the last 20 years, the Arabidopsis Biological Resource Center (ABRC) has collected and developed a series of best-practice protocols, some of which are presented in this chapter. Arabidopsis can be grown in a variety of locations, growth media, and environmental conditions. Some mutant genotypes, natural accessions, and Arabidopsis relatives require strictly controlled growth conditions best provided by growth rooms, chambers, or incubators. Other lines can be grown in less-controlled greenhouse settings. Although the majority of lines can be grown in soil, certain experimental purposes require utilization of sterile solid or liquid growth media. These include the selection of primary transformants, identification of homozygous lethal individuals in a segregating population, or bulking of a large amount of plant material. The importance of controlling, observing, and recording growth conditions is emphasized and appropriate equipment for monitoring these conditions is listed. Proper conditions for seed harvest and preservation, as well as seed quality control procedures, are also described. In addition, plant transformation and genetic crosses, two of the methods that revolutionized Arabidopsis genetics, are discussed.

Using Cell Cycle-Restricted Alleles to Study the Chromatin Dynamics and Functions of the Structural Maintenance of Chromosomes (SMC) Complexes In Vivo.

SMC complexes play fundamental functions in chromosome architecture and organization as well as in DNA replication and repair throughout the cell cycle. The essential nature of the SMC components makes the study of their specific functions challenging. In this chapter, we describe the application of cell cycle tags to S. cerevisiae SMC genes. The cell cycle tags regulate both gene expression and protein degradation, allowing for restriction of the gene of interest to either the S or the G2/M phase. In case of SMC genes, the tags lead to valuable mutants that can bring insights into cell cycle specific essential functions, chromatin binding pattern and functional interactions. Here, we describe the generation of the cell cycle-restricted mutants in diploid and haploid cells and the validation of their functionality with several approaches.

Maternal mitochondrial inheritance in two Fusarium pathogens of prickly ash (Zanthoxylum bungeanum) in northern China.

Mitochondrial inheritance in Fusarium zanthoxyli and F. continuum, two canker-inducing pathogens of prickly ash (Zanthoxylum bungeanum) in northern China, was investigated by genotyping ascospore progeny obtained from laboratory crosses. Polymorphic regions of the mitochondrial genomes (mitogenomes) that contained indels and single-nucleotide polymorphisms (SNPs) were identified via comparative analyses of the complete mitogenomes of the parents used in the intraspecific crosses. A reciprocal genetic cross of F. zanthoxyli NRRL 66714 × NRRL 66285, and a separate cross of F. continuum ♀ NRRL 66286 × â™‚ NRRL 66218, revealed that mitochondria were only inherited from the maternal parent. In addition, the reciprocal cross demonstrated that mitochondrial inheritance is not linked to mating type. Gene order in the circular mitogenomes of the prickly ash pathogens was identical to that previously reported for other fusaria and members of the Hypocreales, except that the TRNL tRNAs were duplicated in F. zanthoxyli NRRL 66714. The genomes contained 14 polypeptide-encoding genes involved in oxidative respiration, one intron-encoded ribosomal protein (rps3) gene, two ribosomal RNA (rRNA) genes, and 26-28 tRNA genes. The F. zanthoxyli mitogenomes were 80.9 and 98.7 kb in length, whereas those of F. continuum were considerably shorter and nearly identical in length at 63.4 kb. The significant differences in mitogenome length were primarily due to variable numbers of introns and open reading frames (ORFs) encoding hypothetical proteins.

eQTL Epistasis - Challenges and Computational Approaches.

The determination of expression quantitative trait loci (eQTL) epistasis - a form of functional interaction between genetic loci that affect gene expression - is an important step toward the thorough understanding of gene regulation. Since gene expression has emerged as an "intermediate" molecular phenotype eQTL epistasis might help to explain the relationship between genotype and higher level organismal phenotypes such as diseases. A characteristic feature of eQTL analysis is the big number of tests required to identify associations between gene expression and genetic loci variability. This problem is aggravated, when epistatic effects between eQTLs are analyzed. In this review, we discuss recent algorithmic approaches for the detection of eQTL epistasis and highlight lessons that can be learned from current methods.