Chromosome-level reference genomes for two strains of Caenorhabditis briggsae: an improved platform for comparative genomics

The publication of the Caenorhabditis briggsae reference genome in 2003 enabled the first comparative genomics studies between C. elegans and C. briggsae, shedding light on the evolution of genome content and structure in the Caenorhabditis genus. However, despite being widely used, the currently available C. briggsae reference genome is substantially less complete and structurally accurate than the C. elegans reference genome. Here, we used high-coverage Oxford Nanopore long-read and chromosome conformation capture data to generate chromosomally resolved reference genomes for two C. briggsae strains: QX1410, a new reference strain closely related to the laboratory AF16 strain, and VX34, a highly divergent strain isolated in China. We also sequenced 99 recombinant inbred lines (RILs) generated from reciprocal crosses between QX1410 and VX34 to create a recombination map and identify chromosomal domains. Additionally, we used both short- and long-read RNA sequencing (RNA-seq) data to generate high-quality gene annotations. By comparing these new reference genomes to the current reference, we reveal that hyper-divergent haplotypes cover large portions of the C. briggsae genome, similar to recent reports in C. elegans and C. tropicalis. We also show that the genomes of selfing Caenorhabditis species have undergone more rearrangement than their outcrossing relatives, which has biased previous estimates of rearrangement rate in Caenorhabditis. These new genomes provide a substantially improved platform for comparative genomics in Caenorhabditis and narrow the gap between the quality of genomic resources available for C. elegans and C. briggsae.

Gene identification and genome annotation in Caenorhabditis briggsae by high throughput 5' RNA end determination

The nematode Caenorhabditis briggsae is routinely used in comparative and evolutionary studies involving its well-known cousin C. elegans. The C. briggsae genome sequence has accelerated research by facilitating the generation of new resources, tools, and functional studies of genes. While substantial progress has been made in predicting genes and start sites, experimental evidence is still lacking in many cases. Here, we report an improved annotation of the C. briggsae genome using the Trans-spliced Exon Coupled RNA End Determination (TEC-RED) technique. In addition to identifying 5' ends of expressed genes, the technique has enabled the discovery of operons and paralogs. Application of TEC-RED yielded 10,243 unique 5' end sequences with matches in the C. briggsae genome. Of these, 6,395 were found to represent 4,252 unique genes along with 362 paralogs and 52 previously unknown exons. The method also identified 493 operons, including 334 that are fully supported by tags. Additionally, two SL1-type operons were discovered. Comparisons with C. elegans revealed that 40% of operons are conserved. Further, we identified 73 novel operons, including 12 that entirely lack orthologs in C. elegans. Among other results, we found that 14 genes are trans-spliced exclusively in C. briggsae compared with C. elegans. Altogether, the data presented here serves as a rich resource to aid biological studies involving C. briggsae. Additionally, this work demonstrates the use of TEC-RED for the first time in a non-elegans nematode and suggests that it could apply to other organisms with a trans-splicing reaction from spliced leader RNA.

Cold survival and its molecular mechanisms in a locally adapted nematode population

Since Darwin, evolutionary biologists have sought to understand the drivers and mechanisms of natural trait diversity. The field advances toward this goal with the discovery of phenotypes that vary in the wild, their relationship to ecology, and their underlying genes. Here, we established resistance to extreme low temperature in the free-living nematode Caenorhabditis briggsae as an ecological and evolutionary model system. We found that C. briggsae strains of temperate origin were strikingly more cold-resistant than those isolated from tropical localities. Transcriptional profiling revealed expression patterns unique to the resistant temperate ecotype, including dozens of genes expressed at high levels even after multiple days of cold-induced physiological slowdown. Mutational analysis validated a role in cold resistance for seven such genes. As the temperate C. briggsae population likely diverged only ~700 years ago from tropical ancestors, our findings highlight a candidate case of very rapid, robust, and genetically complex adaptation, and shed light on the mechanisms at play.

TOXIC ACTION OF COPPER, CADMIUM AND LEAD ON FREE-LIVING SOIL NEMATODES CAENORHABDITIS ELEGANS AND CAENORHABDITIS BRIGGSAE

Heavy metals are one of the most common pollutants in environment. The aim of this work was to test the hypothesis assuming that one of mechanisms of toxic action of copper, cadmium and lead on invertebrates’ organisms is adaptive activation of cholinergic synaptic transmission. In experiments with two free-living soil nematodes, namely Caenorhabditis elegans and Caenorhabditis briggsae, it has been shown that Cu2+, Cd2+ and Pb2+ ions at concentrations of 60 and 120 µM enhanced the negative effects of the nicotinic acetylcholine receptors agonist levamisole on the nematodes’ organisms. Under combined action of levamisole and heavy metals on organisms of C. elegans and C. briggsae the mean time of nematodes paralysis (complete loss of the ability to swim) was reduced. The results of this work show that nematodes C. elegans and C. briggsae can be used as model organisms to study mechanisms of toxic action of heavy metals.

Mitochondrial DNA Variation and Selfish Propagation Following Experimental Bottlenecking in Two Distantly Related Caenorhabditis briggsae Isolates

Understanding mitochondrial DNA (mtDNA) evolution and inheritance has broad implications for animal speciation and human disease models. However, few natural models exist that can simultaneously represent mtDNA transmission bias, mutation, and copy number variation. Certain isolates of the nematode Caenorhabditis briggsae harbor large, naturally-occurring mtDNA deletions of several hundred basepairs affecting the NADH dehydrogenase subunit 5 (nduo-5) gene that can be functionally detrimental. These deletion variants can behave as selfish DNA elements under genetic drift conditions, but whether all of these large deletion variants are transmitted in the same preferential manner remains unclear. In addition, the degree to which transgenerational mtDNA evolution profiles are shared between isolates that differ in their propensity to accumulate the nduo-5 deletion is also unclear. We address these knowledge gaps by experimentally bottlenecking two isolates of C. briggsae with different nduo-5 deletion frequencies for up to 50 generations and performing total DNA sequencing to identify mtDNA variation. We observed multiple mutation profile differences and similarities between C. briggsae isolates, a potentially species-specific pattern of copy number dysregulation, and some evidence for genetic hitchhiking in the deletion-bearing isolate. Our results further support C. briggsae as a practical model for characterizing naturally-occurring mtgenome variation and contribute to the understanding of how mtgenome variation persists in animal populations and how it presents in mitochondrial disease states.

Genome editing in the nematode Caenorhabditis briggsae using the CRISPR/Cas9 system

The CRISPR/Cas system has recently emerged as a powerful tool to engineer the genome of an organism. The system is adopted from bacteria where it confers immunity against invading foreign DNA. This work reports the first successful use of the CRISPR/Cas system in Caenorhabditis briggsae (a cousin of the well-known nematode C. elegans), to generate mutations via non-homologous end joining. We recovered deletion alleles of several conserved genes by microinjecting plasmids that express Cas9 endonuclease and an engineered CRISPR RNA corresponding to the DNA sequence to be cleaved. Evidence for somatic mutations and off-target mutations are also reported. Our approach allows for the generation of loss-of-function mutations in C. briggsae genes thereby facilitating a comparative study of gene function.

Natural Viruses of Caenorhabditis Nematodes

Caenorhabditis elegans has long been a laboratory model organism with no known natural pathogens. In the past ten years, however, natural viruses have been isolated from wild-caught C. elegans (Orsay virus) and its relative Caenorhabditis briggsae (Santeuil virus, Le Blanc virus, and Melnik virus). All are RNA positive-sense viruses related to Nodaviridae; they infect intestinal cells and are horizontally transmitted. The Orsay virus capsid structure has been determined and the virus can be reconstituted by transgenesis of the host. Recent use of the Orsay virus has enabled researchers to identify evolutionarily conserved proviral and antiviral genes that function in nematodes and mammals. These pathways include endocytosis through SID-3 and WASP; a uridylyltransferase that destabilizes viral RNAs by uridylation of their 3′ end; ubiquitin protein modifications and turnover; and the RNA interference pathway, which recognizes and degrades viral RNA.

Evolution of sex ratio through gene loss

The maintenance of males at intermediate frequencies is an important evolutionary problem. Several species ofCaenorhabditisnematodes have evolved a mating system in which selfing hermaphrodites and males coexist. While selfing produces XX hermaphrodites, cross-fertilization produces 50% XO male progeny. Thus, male mating success dictates the sex ratio. Here, we focus on the contribution of themale secreted short(mss) gene family to male mating success, sex ratio, and population growth. Themssfamily is essential for sperm competitiveness in gonochoristic species, but has been lost in parallel in androdioecious species. Using a transgene to restoremssfunction to the androdioeciousCaenorhabditis briggsae,we examined how mating system and population subdivision influence the fitness of themss+genotype. Consistent with theoretical expectations, whenmss+andmss-null (i.e., wild type) genotypes compete,mss+is positively selected in both mixed-mating and strictly outcrossing situations, though more strongly in the latter. Thus, while sexual mode alone affects the fitness ofmss+, it is insufficient to explain its parallel loss. However, in genetically homogenous androdioecious populations,mss+both increases male frequency and depresses population growth. We propose that the lack of inbreeding depression and the strong subdivision that characterize naturalCaenorhabditispopulations impose selection on sex ratio that makes loss ofmssadaptive after self-fertility evolves.

The ebb and flow of heteroplasmy during intra-species hybridization in Caenorhabditis briggsae

ABSTRACTMitochondria are typically maternally inherited. In many species, this transmission pattern is produced by sperm-borne mitochondria being eliminated either from sperm before fertilization or from the embryo after fertilization. In the nematode Caenorhabditis briggsae, repeatedly backcrossing hybrids to genetically diverse males can elicit paternal mitochondrial transmission. Studies of other taxa also suggest that hybridization increases paternal mitochondrial transmission. Thus, hybrid genotypes might disrupt the systems that normally prevent paternal mitochondrial transmission. Given the reliance of a number of genetic analyses on the assumption of purely maternal mitochondrial inheritance, it would be broadly valuable to learn more about the processes embryos employ to prevent sperm-borne mitochondria from persisting in offspring, as well as the circumstances under which paternal transmission might be expected to occur. To quantify the tempo of paternal mitochondrial transmission in hybrids, we assessed the presence of paternal mitotypes in replicate lines at three timepoints spanning fifteen generations. All lines exhibited paternal mitochondrial transmission. However, this heteroplasmy always then resolved to homoplasmy for the maternal mitotype. Additionally, one nuclear locus exhibited allele transmission ratio distortion that might reflect mito-nuclear co-evolution. This study frames the genetic architecture of a hybrid genetic incompatibility that leads to paternal mitochondrial transmission and to a reduction in hybrid fitness.