Chromosome fusions repatterned recombination rate and facilitated reproductive isolation during Pristionchus nematode speciation.

Large-scale genome-structural evolution is common in various organisms. Recent developments in speciation genomics revealed the importance of inversions, whereas the role of other genome-structural rearrangements, including chromosome fusions, have not been well characterized. We study genomic divergence and reproductive isolation of closely related nematodes: the androdioecious (hermaphroditic) model Pristionchus pacificus and its dioecious sister species Pristionchus exspectatus. A chromosome-level genome assembly of P. exspectatus using single-molecule and Hi-C sequencing revealed a chromosome-wide rearrangement relative to P. pacificus. Strikingly, genomic characterization and cytogenetic studies including outgroup species Pristionchus occultus indicated two independent fusions involving the same chromosome, ChrIR, between these related species. Genetic linkage analysis indicated that these fusions altered the chromosome-wide pattern of recombination, resulting in large low-recombination regions that probably facilitated the coevolution between some of the ~14.8% of genes across the entire genomes. Quantitative trait locus analyses for hybrid sterility in all three sexes revealed that major quantitative trait loci mapped to the fused chromosome ChrIR. While abnormal chromosome segregations of the fused chromosome partially explain hybrid female sterility, hybrid-specific recombination that breaks linkage of genes in the low-recombination region was associated with hybrid male sterility. Thus, recent chromosome fusions repatterned recombination rate and drove reproductive isolation during Pristionchus speciation.

The compact genome of Caenorhabditis niphades n. sp., isolated from a wood-boring weevil, Niphades variegatus.

BACKGROUND: The first metazoan genome sequenced, that of Caenorhabditis elegans, has motivated animal genome evolution studies. To date > 50 species from the genus Caenorhabditis have been sequenced, allowing research on genome variation. RESULTS: In the present study, we describe a new gonochoristic species, Caenorhabditis niphades n. sp., previously referred as C. sp. 36, isolated from adult weevils (Niphades variegatus), with whom they appear to be tightly associated during its life cycle. Along with a species description, we sequenced the genome of C. niphades n. sp. and produced a chromosome-level assembly. A genome comparison highlighted that C. niphades n. sp. has the smallest genome (59 Mbp) so far sequenced in the Elegans supergroup, despite being closely related to a species with an exceptionally large genome, C. japonica. CONCLUSIONS: The compact genome of C. niphades n. sp. can serve as a key resource for comparative evolutionary studies of genome and gene number expansions in Caenorhabditis species.

The GATA factor ELT-3 specifies endoderm in Caenorhabditis angaria in an ancestral gene network.

Endoderm specification in Caenorhabditis elegans occurs through a network in which maternally provided SKN-1/Nrf, with additional input from POP-1/TCF, activates the GATA factor cascade MED-1,2→END-1,3→ELT-2,7. Orthologues of the MED, END and ELT-7 factors are found only among nematodes closely related to C. elegans, raising the question of how gut is specified in their absence in more distant species in the genus. We find that the C. angaria, C. portoensis and C. monodelphis orthologues of the GATA factor gene elt-3 are expressed in the early E lineage, just before their elt-2 orthologues. In C. angaria, Can-pop-1(RNAi), Can-elt-3(RNAi) and a Can-elt-3 null mutation result in a penetrant 'gutless' phenotype. Can-pop-1 is necessary for Can-elt-3 activation, showing that it acts upstream. Forced early E lineage expression of Can-elt-3 in C. elegans can direct the expression of a Can-elt-2 transgene and rescue an elt-7 end-1 end-3; elt-2 quadruple mutant strain to viability. Our results demonstrate an ancestral mechanism for gut specification and differentiation in Caenorhabditis involving a simpler POP-1→ELT-3→ELT-2 gene network.

Possible stochastic sex determination in Bursaphelenchus nematodes.

Sex determination mechanisms evolve surprisingly rapidly, yet little is known in the large nematode phylum other than for Caenorhabditis elegans, which relies on chromosomal XX-XO sex determination and a dosage compensation mechanism. Here we analyze by sex-specific genome sequencing and genetic analysis sex determination in two fungal feeding/plant-parasitic Bursaphelenchus nematodes and find that their sex differentiation is more likely triggered by random, epigenetic regulation than by more well-known mechanisms of chromosomal or environmental sex determination. There is no detectable difference in male and female chromosomes, nor any linkage to sexual phenotype. Moreover, the protein sets of these nematodes lack genes involved in X chromosome dosage counting or compensation. By contrast, our genetic screen for sex differentiation mutants identifies a Bursaphelenchus ortholog of tra-1, the major output of the C. elegans sex determination cascade. Nematode sex determination pathways might have evolved by "bottom-up" accretion from the most downstream regulator, tra-1.

Genome of the fatal tapeworm Sparganum proliferum uncovers mechanisms for cryptic life cycle and aberrant larval proliferation.

The cryptic parasite Sparganum proliferum proliferates in humans and invades tissues and organs. Only scattered cases have been reported, but S. proliferum infection is always fatal. However, S. proliferum's phylogeny and life cycle remain enigmatic. To investigate the phylogenetic relationships between S. proliferum and other cestode species, and to examine the mechanisms underlying pathogenicity, we sequenced the entire genomes of S. proliferum and a closely related non-life-threatening tapeworm Spirometra erinaceieuropaei. Additionally, we performed larvae transcriptome analyses of S. proliferum plerocercoid to identify genes involved in asexual reproduction in the host. The genome sequences confirmed that the S. proliferum has experienced a clearly distinct evolutionary history from S. erinaceieuropaei. Moreover, we found that nonordinal extracellular matrix coordination allows asexual reproduction in the host, and loss of sexual maturity in S. proliferum are responsible for its fatal pathogenicity to humans. Our high-quality reference genome sequences should be valuable for future studies of pseudophyllidean tapeworm biology and parasitism.

Additional description and genome analyses of Caenorhabditis auriculariae representing the basal lineage of genus Caenorhabditis.

Caenorhabditis auriculariae, which was morphologically described in 1999, was re-isolated from a Platydema mushroom-associated beetle. Based on the re-isolated materials, some morphological characteristics were re-examined and ascribed to the species. In addition, to clarify phylogenetic relationships with other Caenorhabditis species and biological features of the nematode, the whole genome was sequenced and assembled into 109.5 Mb with 16,279 predicted protein-coding genes. Molecular phylogenetic analyses based on ribosomal RNA and 269 single-copy genes revealed the species is closely related to C. sonorae and C. monodelphis placing them at the most basal clade of the genus. C. auriculariae has morphological characteristics clearly differed from those two species and harbours a number of species-specific gene families, indicating its usefulness as a new outgroup species for Caenorhabditis evolutionary studies. A comparison of carbohydrate-active enzyme (CAZy) repertoires in genomes, which we found useful to speculate about the lifestyle of Caenorhabditis nematodes, suggested that C. auriculariae likely has a life-cycle with tight-association with insects.

Metolazone upregulates mitochondrial chaperones and extends lifespan in Caenorhabditis elegans.

Accumulating studies have argued that the mitochondrial unfolded protein response (UPRmt) is a mitochondrial stress response that promotes longevity in model organisms. In the present study, we screened an off-patent drug library to identify compounds that activate UPRmt using a mitochondrial chaperone hsp-6::GFP reporter system in Caenorhabditis elegans. Metolazone, a diuretic primarily used to treat congestive heart failure and high blood pressure, was identified as a prominent hit as it upregulated hsp-6::GFP and not the endoplasmic reticulum chaperone hsp-4::GFP. Furthermore, metolazone specifically induced the expression of mitochondrial chaperones in the HeLa cell line. Metolazone also extended the lifespan of worms in a atfs-1 and ubl-5-dependent manner. Notably, metolazone failed to increase lifespan in worms with knocked-down nkcc-1. These results suggested that metolazone activates the UPRmt across species and prolongs the lifespan of C. elegans.

Telomere-to-Telomere Genome Assembly of Bursaphelenchus okinawaensis Strain SH1.

Bursaphelenchus okinawaensis is a self-fertilizing, hermaphroditic, fungus-feeding nematode used as a laboratory model for the genus Bursaphelenchus, which includes the important pathogen Bursaphelenchus xylophilus Here, we report the nearly complete genome sequence of B. okinawaensis The 70-Mbp assembly contained six scaffolds (>11 Mbp each) with telomere repeats on their ends, indicating complete chromosomes.

Nearly Complete Genome Assembly of the Pinewood Nematode Bursaphelenchus xylophilus Strain Ka4C1.

Bursaphelenchus xylophilus has been destroying pine forests in East Asia and western Europe. Here, we report its nearly complete genomic sequence containing five ∼12-Mb scaffolds and one ∼15-Mb scaffold representing six chromosomes. Large repeat regions that were previously unidentified are now reasonably integrated, particularly in the ∼15-Mb scaffold.

Real-time nanodiamond thermometry probing in vivo thermogenic responses.

Real-time temperature monitoring inside living organisms provides a direct measure of their biological activities. However, it is challenging to reduce the size of biocompatible thermometers down to submicrometers, despite their potential applications for the thermal imaging of subtissue structures with single-cell resolution. Here, using quantum nanothermometers based on optically accessible electron spins in nanodiamonds, we demonstrate in vivo real-time temperature monitoring inside Caenorhabditis elegans worms. We developed a microscope system that integrates a quick-docking sample chamber, particle tracking, and an error correction filter for temperature monitoring of mobile nanodiamonds inside live adult worms with a precision of ±0.22°C. With this system, we determined temperature increases based on the worms' thermogenic responses during the chemical stimuli of mitochondrial uncouplers. Our technique demonstrates the submicrometer localization of temperature information in living animals and direct identification of their pharmacological thermogenesis, which may allow for quantification of their biological activities based on temperature.

daf-16/FOXO isoform b in AIY neurons is involved in low preference for Bifidobacterium infantis in Caenorhabditis elegans.

The neural and molecular mechanisms underlying food preference have been poorly understood. We previously showed that Bifidobacterium infantis (B. infantis), a well-known probiotic bacterium, extends the lifespan of Caenorhabditis elegans (C. elegans) compared with a standard food, Escherichia coli (E. coli) OP50. In this study, we characterized C. elegans behavior against B. infantis and examined the neural and molecular mechanisms governing that behavior. The majority of the wild-type animals were outside of the B. infantis lawn 10 min after transfer. Although worms did not prefer B. infantis compared to E. coli OP50, they preferred the B. infantis lawn over a lawn containing M9 buffer alone, in which there was no food. Mutant analyses suggested that leaving the B. infantis lawn required daf-16/FOXO. Isoform-specific mutant phenotypes suggested that daf-16 isoform b seemed to be associated with leaving. Genetic rescue experiments demonstrated that the function of daf-16b in AIY interneurons was involved in leaving the B. infantis lawn. The daf-18/PTEN mutants were also defective in leaving. In conclusion, C. elegans showed a low preference for B. infantis, and daf-16b in AIY interneurons and daf-18 had roles in leaving B. infantis.

Toll-like receptor homolog TOL-1 regulates Bifidobacterium infantis-elicited longevity and behavior in Caenorhabditis elegans.

Bifidobacterium infantis, a Gram-positive bacterium, is one of the commonly used probiotics. We previously showed that B. infantis modified host defense systems and extended the lifespan of the nematode Caenorhabditis elegans. In the present study, we showed that the lifespan extension caused by B. infantis was enhanced in animals having a mutation in the tol-1 gene that encodes the sole C. elegans homolog of Toll-like receptors (TLRs). Meanwhile, lifespan increased by other probiotic bacteria, such as Bacillus subtilis or Clostridium butyricum, was not affected in the tol-1 mutant animals. A microarray analysis revealed that the expression of innate immune response-related genes was significantly increased in the tol-1 mutant. Worms with the tol-1 mutation exhibited reduced leaving behavior from the B. infantis lawn, while canonical downstream factors trf-1/TRAF and ikb-1/IκB appeared to not be involved. In conclusion, C. elegans tol-1/TLR regulates B. infantis-induced longevity and also regulates behavior against B. infantis.

The small GTPase ARF-1.2 is a regulator of unicellular tube formation in Caenorhabditis elegans.

The membrane trafficking events that regulate unicellular tube formation and maintenance are not well understood. Here, using an RNAi screen, we identified the small GTPase ARF1 homolog ARF-1.2 as a regulator of excretory tube formation in Caenorhabditis elegans. RNAi-mediated knockdown and knockout of the arf-1.2 gene resulted in the formation of large intracellular vacuoles at the growth sites (varicosities) of the excretory canals. arf-1.2 mutant animals were sensitive to hyperosmotic conditions. arf-1.2 RNAi affected the localization of the anion transporter SULP-8, which is expressed in the basal plasma membrane of the excretory canals, but did not affect the expression of SULP-4, which is expressed in the apical membrane. The phenotype of arf-1.2 mutants was suppressed by mutation of the small Rho GTPase CDC-42, a regulator of apical/basal traffic balance. These results suggest that ARF-1.2 plays an essential role in basal membrane traffic to regulate the formation of the unicellular excretory tube.

Clostridium butyricum MIYAIRI 588 Increases the Lifespan and Multiple-Stress Resistance of Caenorhabditis elegans.

Clostridium butyricum MIYAIRI 588 (CBM 588), one of the probiotic bacterial strains used for humans and domestic animals, has been reported to exert a variety of beneficial health effects. The effect of this probiotic on lifespan, however, is unknown. In the present study, we investigated the effect of CBM 588 on lifespan and multiple-stress resistance using Caenorhabditis elegans as a model animal. When adult C. elegans were fed a standard diet of Escherichia coli OP50 or CBM 588, the lifespan of the animals fed CBM 588 was significantly longer than that of animals fed OP50. In addition, the animals fed CBM588 exhibited higher locomotion at every age tested. Moreover, the worms fed CBM 588 were more resistant to certain stressors, including infections with pathogenic bacteria, UV irradiation, and the metal stressor Cu2+. CBM 588 failed to extend the lifespan of the daf-2/insulin-like receptor, daf-16/FOXO and skn-1/Nrf2 mutants. In conclusion, CBM 588 extends the lifespan of C. elegans probably through regulation of the insulin/IGF-1 signaling (IIS) pathway and the Nrf2 transcription factor, and CBM 588 improves resistance to several stressors in C. elegans.

Caenorhabditis elegans homologue of Prox1/Prospero is expressed in the glia and is required for sensory behavior and cold tolerance.

The Caenorhabditis elegans (C. elegans) amphid sensory organ contains only 4 glia-like cells and 24 sensory neurons, providing a simple model for analyzing glia or neuron-glia interactions. To better characterize glial development and function, we carried out RNA interference screening for transcription factors that regulate the expression of an amphid sheath glial cell marker and identified pros-1, which encodes a homeodomain transcription factor homologous to Drosophila prospero/mammalian Prox1, as a positive regulator. The functional PROS-1::EGFP fusion protein was localized in the nuclei of the glia and the excretory cell but not in the amphid sensory neurons. pros-1 deletion mutants exhibited larval lethality, and rescue experiments showed that pros-1 and human Prox1 transgenes were able to rescue the larval lethal phenotype, suggesting that pros-1 is a functional homologue of mammalian Prox1, at least partially. We further found that the structure and functions of sensory neurons, such as the morphology of sensory endings, sensory behavior and sensory-mediated cold tolerance, appeared to be affected by the pros-1 RNAi. Together, our results show that the C. elegans PROS-1 is a transcriptional regulator in the glia but is involved not only in sensory behavior but also in sensory-mediated physiological tolerance.