Transcriptional profiles in Strongyloides stercoralis males reveal deviations from the Caenorhabditis sex determination model.

The human and canine parasitic nematode Strongyloides stercoralis utilizes an XX/XO sex determination system, with parasitic females reproducing by mitotic parthenogenesis and free-living males and females reproducing sexually. However, the genes controlling S. stercoralis sex determination and male development are unknown. We observed precocious development of rhabditiform males in permissive hosts treated with corticosteroids, suggesting that steroid hormones can regulate male development. To examine differences in transcript abundance between free-living adult males and other developmental stages, we utilized RNA-Seq. We found two clusters of S. stercoralis-specific genes encoding predicted transmembrane proteins that are only expressed in free-living males. We additionally identified homologs of several genes important for sex determination in Caenorhabditis species, including mab-3, tra-1, fem-2, and sex-1, which may have similar functions. However, we identified three paralogs of gld-1; Ss-qki-1 transcripts were highly abundant in adult males, while Ss-qki-2 and Ss-qki-3 transcripts were highly abundant in adult females. We also identified paralogs of pumilio domain-containing proteins with sex-specific transcripts. Intriguingly, her-1 appears to have been lost in several parasite lineages, and we were unable to identify homologs of tra-2 outside of Caenorhabditis species. Together, our data suggest that different mechanisms control male development in S. stercoralis and Caenorhabditis species.

Planarian EGF repeat-containing genes megf6 and hemicentin are required to restrict the stem cell compartment.

The extracellular matrix (ECM) is important for maintaining the boundaries between tissues. This role is particularly critical in the stem cell niche, as pre-neoplastic or cancerous stem cells must pass these boundaries in order to invade into the surrounding tissue. Here, we examine the role of the ECM as a regulator of the stem cell compartment in the planarian Schmidtea mediterranea, a highly regenerative, long-lived organism with a large population of adult stem cells. We identify two EGF repeat-containing genes, megf6 and hemicentin, with identical knockdown phenotypes. We find that megf6 and hemicentin are needed to maintain the structure of the basal lamina, and in the absence of either gene, pluripotent stem cells migrate ectopically outside of their compartment and hyper-proliferate, causing lesions in the body wall muscle. These muscle lesions and ectopic stem cells are also associated with ectopic gut branches, which protrude from the normal gut towards the dorsal side of the animal. Interestingly, both megf6 and hemicentin knockdown worms are capable of regenerating tissue free of both muscle lesions and ectopic cells, indicating that these genes are dispensable for regeneration. These results provide insight into the role of planarian ECM in restricting the stem cell compartment, and suggest that signals within the compartment may act to suppress stem cell hyperproliferation.

Schistosoma mansoni: Off-target analyses using nonspecific double-stranded RNAs as control for RNAi experiments in schistosomula.

RNA interference is a well established and widely used reverse genetic tool available for gene functional studies in trematodes. This technique requires the use of nonrelevant double-stranded RNA as control. However, several authors have reported inconsistencies associated with RNAi. We used RNASeq to analyze genes affected by nonspecific dsRNA exposure. We found only few genes presenting altered expression in schistosomula exposed to GFP or mCherry nonspecific-dsRNAs, most of them encoding uncharacterized proteins. Correlation analysis revealed that there are more differences among biological replicates, than due to treatment with nonspecific controls. These observations are of key relevance to other RNAi gene function assessment in other organisms.

The complete mitochondrial genome of Tetrastemma olgarum (Nemertea: Hoplonemertea).

The complete mitochondrial genome (mitogenome) of Tetrastemma olgarum is sequenced. It is 14,580 bp in length and contains 37 genes typical for metazoan mitogenomes. The gene order is identical to that of the previously published Hoplonemertea mitogenomes. All genes are encoded on the heavy strand except for trnT and trnP. The coding strand is AT-rich, accounting for 69.2% of overall nucleotide composition.

Mixed bioengineering-chemical synthesis approach for the efficient preparation of Δ7-dafachronic acid.

Combining bioengineering with chemical synthesis has enabled an efficient method for producing Δ7-dafachronic acid, a steroidal hormone associated with nematode germline longevity. Saccharomyces cerevisiae was engineered to produce 7,24-cholestadienol, a convenient starting material for a four-step synthesis of Δ7-dafachronic acid.

Identification of novel target genes for safer and more specific control of root-knot nematodes from a pan-genome mining.

Root-knot nematodes are globally the most aggressive and damaging plant-parasitic nematodes. Chemical nematicides have so far constituted the most efficient control measures against these agricultural pests. Because of their toxicity for the environment and danger for human health, these nematicides have now been banned from use. Consequently, new and more specific control means, safe for the environment and human health, are urgently needed to avoid worldwide proliferation of these devastating plant-parasites. Mining the genomes of root-knot nematodes through an evolutionary and comparative genomics approach, we identified and analyzed 15,952 nematode genes conserved in genomes of plant-damaging species but absent from non target genomes of chordates, plants, annelids, insect pollinators and mollusks. Functional annotation of the corresponding proteins revealed a relative abundance of putative transcription factors in this parasite-specific set compared to whole proteomes of root-knot nematodes. This may point to important and specific regulators of genes involved in parasitism. Because these nematodes are known to secrete effector proteins in planta, essential for parasitism, we searched and identified 993 such effector-like proteins absent from non-target species. Aiming at identifying novel targets for the development of future control methods, we biologically tested the effect of inactivation of the corresponding genes through RNA interference. A total of 15 novel effector-like proteins and one putative transcription factor compatible with the design of siRNAs were present as non-redundant genes and had transcriptional support in the model root-knot nematode Meloidogyne incognita. Infestation assays with siRNA-treated M. incognita on tomato plants showed significant and reproducible reduction of the infestation for 12 of the 16 tested genes compared to control nematodes. These 12 novel genes, showing efficient reduction of parasitism when silenced, constitute promising targets for the development of more specific and safer control means.

Identification and characterization of a Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae) thermotolerance-Related Gene: Bx-HSP90.

Temperatures directly influence the distribution and intensity of pine wilt disease caused by the pine wood nematode, Bursaphelenchus xylophilus. To date, however, little is known about the causation and mechanism of this influence. The molecular chaperone HSP90 is a key component that contributes to survival in the abiotic stress response. In this study, we investigated the relationship between the survival of B. xylophilus and the functionality of the HSP90 gene. Bx-HSP90 was cloned from a suppression subtractive hybridization library. In situ mRNA hybridization showed that Bx-HSP90 was constitutively expressed in response to all of the temperatures tested, and RT-PCR indicated that all of the temperatures could induce Bx-HSP90 transcription, with the highest transcript level detected at 30 °C. The suppression of the Bx-HSP90 transcript by RNA interference led to a 25% reduction in the number of nematodes at 30 °C after 44 h. Sharp declines in the survival of the RNAi-treated nematodes were observed after 8 days at 25 °C, 48 h at 30 °C and 24 h at 35 °C. Both heat shock and the knockdown of Bx-HSP90 hindered the growth of the B. xylophilus populations. The results indicate that Bx-HSP90 is essential for the survival of B. xylophilus, confirming the thermoregulatory function of the gene, and delineate the timeframe and temperature range within which the gene function occurs.

Genome-wide analysis of germline signaling genes regulating longevity and innate immunity in the nematode Pristionchus pacificus.

Removal of the reproductive system of many animals including fish, flies, nematodes, mice and humans can increase lifespan through mechanisms largely unknown. The abrogation of the germline in Caenorhabditis elegans increases longevity by 60% due to a signal emitted from the somatic gonad. Apart from increased longevity, germline-less C. elegans is also resistant to other environmental stressors such as feeding on bacterial pathogens. However, the evolutionary conservation of this pathogen resistance, its genetic basis and an understanding of genes involved in producing this extraordinary survival phenotype are currently unknown. To study these evolutionary aspects we used the necromenic nematode Pristionchus pacificus, which is a genetic model system used in comparison to C. elegans. By ablation of germline precursor cells and subsequent feeding on the pathogen Serratia marcescens we discovered that P. pacificus shows remarkable resistance to bacterial pathogens and that this response is evolutionarily conserved across the Genus Pristionchus. To gain a mechanistic understanding of the increased resistance to bacterial pathogens and longevity in germline-ablated P. pacificus we used whole genome microarrays to profile the transcriptional response comparing germline ablated versus un-ablated animals when fed S. marcescens. We show that lipid metabolism, maintenance of the proteasome, insulin signaling and nuclear pore complexes are essential for germline deficient phenotypes with more than 3,300 genes being differentially expressed. In contrast, gene expression of germline-less P. pacificus on E. coli (longevity) and S. marcescens (immunity) is very similar with only 244 genes differentially expressed indicating that longevity is due to abundant gene expression also involved in immunity. By testing existing mutants of Ppa-DAF-16/FOXO and the nuclear hormone receptor Ppa-DAF-12 we show a conserved function of both genes in resistance to bacterial pathogens and longevity. This is the first study to show that the influence of the reproductive system on extending lifespan and innate immunity is conserved in evolution.

Genomic organization of selected genes in the small monogonont rotifer, Brachionus koreanus.

Information of genome structure with its size variation may provide important clues for evolutionary processes at lower taxon level in eukaryotes. Here, we analyzed the compact genome structure of the monogonont rotifer, Brachionus koreanus in the light of transphyletic genome comparison and economic genome usage. To confirm the genome compactness of B. koreanus, we compared the genomic structure of several selected genes with those of human and pufferfish. For example, one of the large genes, DNA-dependent protein kinase (DNA-PK) with dimeric protein Ku70 and Ku80, showed high similarity, even though genomic DNA lengths were quite different. The replication protein As (RPAs) as a heterotrimeric protein also showed a compact genomic structure including all the essential domains and motifs in B. koreanus. Regarding transmembrane protein-containing genes, the B. koreanus P-glycoprotein (P-gp) showed exactly the same topology of the TM domain compared to those of human and pufferfish, even though it had a compact genome structure. In addition, the gene structure of an inducible repair enzyme O(6)-methylguanine DNA methyltransferase (O(6)-MGMT) of B. koreanus showed the highest compactness among the genes tested. The objective of this report is to evaluate the potential for whole genome sequencing and functional genomic research using the monogonont rotifer B. koreanus as a non-model organism that plays important roles in aquatic food-webs. Subsequently, we discussed possible reasons for compact genome structures as well as small and fewer introns from several perspectives. We conclude that the small size genome of B. koreanus would make this species potentially useful for comparative genome structure analysis of non-model species through whole genome sequencing and genetic mapping.

Comparative transcriptomics of two pathogenic pinewood nematodes yields insights into parasitic adaptation to life on pine hosts.

Bursaphelenchus xylophilus and Bursaphelenchus mucronatus are migratory endoparasitic nematodes that live in pine trees. To gain insight into their molecular similarities and differences, transcriptomes of the two nematodes were analysed. A total of 23,765 and 21,782 contigs (>300 bp) were obtained from B. xylophilus and B. mucronatus, respectively. More than 80% of the contigs could map to each other's transcriptome reciprocally. A total of 23,467 and 21,370 Open Reading Frames were predicted, respectively. Besides those known parasitism-related proteins, six new venom allergen-like proteins (VAPs) were found, which were not homologous to known VAPs. Enzymes involved in xenobiotic biodegradation were abundant in the two transcriptomes based on KEGG functional annotation. Metabolism of xenobiotics by cytochrome P450 comprised the main detoxification pathways. The mRNA expression levels of detoxification genes in nematodes living in the host were higher than those in nematodes feeding on fungus. However, there were fewer enzymes involved in the α-pinene degradation. Our results indicate that the two pinewood nematodes have evolved similar molecular mechanisms to adapt to life on pine hosts.

Ups and downs of RNA interference in parasitic nematodes.

RNA interference (RNAi) is widely used in Caenorhabiditis elegans to identify essential gene function. In parasitic nematodes RNAi has been reported to result in transcript knockdown of some target genes, but not others, thus limiting its use as a potential functional genomics tool. We recently extended work in Haemonchus contortus to examine why only some genes seem to be susceptible to RNAi and to test RNAi effects in vivo. Here we review our findings, which suggest that site of gene expression influences silencing. This most likely reflects limited uptake of dsRNA from the environment, a phenomenon also observed in other free-living nematodes. We discuss new technologies to improve dsRNA delivery, such as nanoparticles being developed for therapeutic siRNA delivery, and methods to monitor RNAi effects. Alternative approaches will be important in progressing the application of RNAi to identify essential gene function in parasitic nematodes.

Caenorhabditis elegans life span studies: the challenge of maintaining synchronous cohorts.

Due to its many advantages, the nematode worm Caenorhabditis elegans (C. elegans) is commonly employed as a convenient model for aging studies as well as for testing life span effects of chemical compounds. However, some challenges exist in the context of such life span studies, particularly in relation to generation and maintenance of synchronized cohorts, and these challenges are not always fully appreciated. Here we discuss the impact of incomplete control of nematode proliferation on life span studies and suggest some solutions to minimize these artefacts.

Symmetry breaking and polarization of the C. elegans zygote by the polarity protein PAR-2.

Polarization of the C. elegans zygote is initiated by ECT-2-dependent cortical flows, which mobilize the anterior PAR proteins (PAR-3, PAR-6 and PKC-3) away from the future posterior end of the embryo marked by the sperm centrosome. Here, we demonstrate the existence of a second, parallel and redundant pathway that can polarize the zygote in the absence of ECT-2-dependent cortical flows. This second pathway depends on the polarity protein PAR-2. We show that PAR-2 localizes to the cortex nearest the sperm centrosome even in the absence of cortical flows. Once on the cortex, PAR-2 antagonizes PAR-3-dependent recruitment of myosin, creating myosin flows that transport the anterior PAR complex away from PAR-2 in a positive-feedback loop. We propose that polarity in the C. elegans zygote is initiated by redundant ECT-2- and PAR-2-dependent mechanisms that lower PAR-3 levels locally, triggering a positive-feedback loop that polarizes the entire cortex.

Regulation of metabolism in Caenorhabditis elegans longevity.

The nematode Caenorhabditis elegans is a favorite model for the study of aging. A wealth of genetic and genomic studies show that metabolic regulation is a hallmark of life-span modulation. A recent study in BMC Biology identifying metabolic signatures for longevity suggests that amino-acid pools may be important in longevity.

RNAi screens to identify components of gene networks that modulate aging in Caenorhabditis elegans.

Our understanding of the genetic mechanisms of organismal aging has advanced dramatically during the past two decades. With the development of large-scale RNAi screens, the last few years saw the remarkable identifications of hundreds of new longevity genes in the roundworm Caenorhabditis elegans. The various RNAi screens revealed many biological pathways previously unknown to be related to aging. In this review, we focus on findings from the recent large-scale RNAi longevity screens, and discuss insights they have provided into the complex biological process of aging and considerations of the RNAi technology will continue to have on the future development of the aging field.

New tools for investigating the comparative biology of Caenorhabditis briggsae and C. elegans.

Comparative studies of Caenorhabditis briggsae and C. elegans have provided insights into gene function and developmental control in both organisms. C. elegans is a well developed model organism with a variety of molecular and genetic tools to study gene functions. In contrast, there are only very limited tools available for its closest relative, C. briggsae. To take advantage of the full potential of this comparative approach, we have developed several genetic and molecular tools to facilitate functional analysis in C. briggsae. First, we designed and implemented an SNP-based oligonucleotide microarray for rapid mapping of genetic mutants in C. briggsae. Second, we generated a mutagenized frozen library to permit the isolation of targeted deletions and used the library to recover a deletion mutant of cbr-unc-119 for use as a transgenic marker. Third, we used the cbr-unc-119 mutant in ballistic transformation and generated fluorescently labeled strains that allow automated lineaging and cellular resolution expression analysis. Finally, we demonstrated the potential of automated lineaging by profiling expression of egl-5, hlh-1, and pha-4 at cellular resolution and by detailed phenotyping of the perturbations on the Wnt signaling pathway. These additions to the experimental toolkit for C. briggsae should greatly increase its utility in comparative studies with C. elegans. With the emerging sequence of nematode species more closely related to C. briggsae, these tools may open novel avenues of experimentation in C. briggsae itself.

Transgenesis in Caenorhabditis elegans.

Two efficient strategies have been developed and are widely used for the genetic transformation of the nematode Caenorhabditis elegans, DNA microinjection, and DNA-coated microparticle bombardment. Both methodologies facilitate the delivery of exogenous DNA into the developing oocytes of adult hermaphrodite animals, which then generate transgenic worms among their progeny. Although both approaches share the common underlying principle of introducing foreign DNA into the germline of C. elegans, they offer distinct transformation outcomes. In this chapter, we present DNA microinjection and bombardment methods for transgenesis in C. elegans and provide time-tested procedures for their implementation. We also discuss their relative advantages as well as their limitations and evaluate their potential for a range of applications.

Germline expression influences operon organization in the Caenorhabditis elegans genome.

Operons are found across multiple kingdoms and phyla, from prokaryotes to chordates. In the nematode Caenorhabditis elegans, the genome contains >1000 operons that compose approximately 15% of the protein-coding genes. However, determination of the force(s) promoting the origin and maintenance of operons in C. elegans has proved elusive. Compared to bacterial operons, genes within a C. elegans operon often show poor coexpression and only sometimes encode proteins with related functions. Using analysis of microarray and large-scale in situ hybridization data, we demonstrate that almost all operon-encoded genes are expressed in germline tissue. However, genes expressed during spermatogenesis are excluded from operons. Operons group together along chromosomes in local clusters that also contain monocistronic germline-expressed genes. Additionally, germline expression of genes in operons is largely independent of the molecular function of the encoded proteins. These analyses demonstrate that mechanisms governing germline gene expression influence operon origination and/or maintenance. Thus, gene expression in a specific tissue can have profound effects on the evolution of genome organization.

Information-based methods for predicting gene function from systematic gene knock-downs.

BACKGROUND: The rapid annotation of genes on a genome-wide scale is now possible for several organisms using high-throughput RNA interference assays to knock down the expression of a specific gene. To date, dozens of RNA interference phenotypes have been recorded for the nematode Caenorhabditis elegans. Although previous studies have demonstrated the merit of using knock-down phenotypes to predict gene function, it is unclear how the data can be used most effectively. An open question is how to optimally make use of phenotypic observations, possibly in combination with other functional genomics datasets, to identify genes that share a common role. RESULTS: We compared several methods for detecting gene-gene functional similarity from phenotypic knock-down profiles. We found that information-based measures, which explicitly incorporate a phenotype's genomic frequency when calculating gene-gene similarity, outperform non-information-based methods. We report the presence of newly predicted modules identified from an integrated functional network containing phenotypic congruency links derived from an information-based measure. One such module is a set of genes predicted to play a role in regulating body morphology based on their multiply-supported interactions with members of the TGF-beta signaling pathway. CONCLUSION: Information-based metrics significantly improve the comparison of phenotypic knock-down profiles, based upon their ability to enhance gene function prediction and identify novel functional modules.

Age-related behaviors have distinct transcriptional profiles in Caenorhabditis elegans.

There has been a great deal of interest in identifying potential biomarkers of aging. Biomarkers of aging would be useful to predict potential vulnerabilities in an individual that may arise well before they are chronologically expected, due to idiosyncratic aging rates that occur between individuals. Prior attempts to identify biomarkers of aging have often relied on the comparisons of long-lived animals to a wild-type control. However, the effect of interventions in model systems that prolong lifespan (such as single gene mutations or caloric restriction) can sometimes be difficult to interpret due to the manipulation itself having multiple unforeseen consequences on physiology, unrelated to aging itself. The search for predictive biomarkers of aging therefore is problematic, and the identification of metrics that can be used to predict either physiological or chronological age would be of great value. One methodology that has been used to identify biomarkers for numerous pathologies is gene expression profiling. Here, we report whole-genome expression profiles of individual wild-type Caenorhabditis elegans covering the entire wild-type nematode lifespan. Individual nematodes were scored for either age-related behavioral phenotypes, or survival, and then subsequently associated with their respective gene expression profiles. This facilitated the identification of transcriptional profiles that were highly associated with either physiological or chronological age. Overall, our approach serves as a paradigm for identifying potential biomarkers of aging in higher organisms that can be repeatedly sampled throughout their lifespan.