Reproductive Span of Caenorhabditis Elegans is Extended by Microbacterium Sp.

The reproductive span (RS) of organisms could be affected by different factors during their lifetime. In the model nematode, Caenorhabditis elegans, RS is affected by both genetic and environmental factors. However, none of the factors identified so far were related to environmental bacteria, which may incidentally appear anywhere in the habitats of C. elegans. We aimed to find environmental bacteria that could affect the RS of C. elegans and related species. We tested 109 bacterial isolates and found that Microbacterium sp. CFBb37 increased the RS and lifespan of C. elegans but reduced its brood size. We studied the effect of M. sp. CFBb37 on the RS of Caenorhabditis briggsae, Caenorhabditis tropicalis, and another Rhabditidae family species, Protorhabditis sp., and found similar trends of RS extension in all three cases, suggesting that this bacterial species may induce the extension of RS broadly among Caenorhabditis species and possibly for many other Rhabditidae. This work will facilitate future research on the mechanism underlying the bacterial extension of RS of nematodes and possibly other animals.     

Evolution of susceptibility to ingested double-stranded RNAs in Caenorhabditis nematodes

Background

The nematode Caenorhabditis elegans is able to take up external double-stranded RNAs (dsRNAs) and mount an RNA interference response, leading to the inactivation of specific gene expression. The uptake of ingested dsRNAs into intestinal cells has been shown to require the SID-2 transmembrane protein in C. elegans. By contrast, C. briggsae was shown to be naturally insensitive to ingested dsRNAs, yet could be rendered sensitive by transgenesis with the C. elegans sid-2 gene. Here we aimed to elucidate the evolution of the susceptibility to external RNAi in the Caenorhabditis genus.

Principal Findings

We study the sensitivity of many new species of Caenorhabditis to ingested dsRNAs matching a conserved actin gene sequence from the nematode Oscheius tipulae. We find ample variation in the Caenorhabditis genus in the ability to mount an RNAi response. We map this sensitivity onto a phylogenetic tree, and show that sensitivity or insensitivity have evolved convergently several times. We uncover several evolutionary losses in sensitivity, which may have occurred through distinct mechanisms. We could render C. remanei and C. briggsae sensitive to ingested dsRNAs by transgenesis of the Cel-sid-2 gene. We thus provide tools for RNA interference studies in these species. We also show that transgenesis by injection is possible in many Caenorhabditis species.

Conclusions

The ability of animals to take up dsRNAs or to respond to them by gene inactivation is under rapid evolution in the Caenorhabditis genus. This study provides a framework and tools to use RNA interference and transgenesis in various Caenorhabditis species for further comparative and evolutionary studies.     

REPRODUCTIVE ISOLATION IN RHABDITIDAE (NEMATODA: SECERNENTEA); MECHANISMS THAT ISOLATE SIX SPECIES OF THREE GENERA

We have attempted interspecific hybridizations among six species of rhabditid nematodes: Caenorhabditis elegans, Caenorhabditis briggsae, Caenorhabditis remanei, Caenorhabditis sp. v, Rhabditis sp., and Pelodera teres. Copulation was observed in all crosses between Caenorhabditis species; however, none resulted in the generation of stable hybrid populations. No copulation was observed in crosses between Caenorhabditis males and Rhabditis or Pelodera females, even when congeneric females were present, suggesting that Caenorhabditis males are able to selectively recognize congeneric females by a short-range stimulus. All pairwise combinations of Caenorhabditis species were isolated to some degree by gametic mechanisms; 7 of 12 combinations were cross infertile and 5 of 12 were cross-fertile but had low brood sizes. In cross-fertile combinations, most hybrid embryos were inviable and arrested prior to gastrulation. Only in crosses of C. briggsae males to C. sp. v females did any hybrids survive embryogenesis. Most of these C. briggsae/C. sp. v hybrids arrested during larval development, and the few that reached adulthood invariably were female. These results are consistent with the presence of at least two lethal factors in the C. briggsae-C. sp. v combination: a maternal lethal factor in the cytoplasm of C. briggsae and a recessive lethal factor on the X chromosome of C. sp. v.     

Potential regulatory elements of nematode vitellogenin genes revealed by interspecies sequence comparison

Summary The nematode,Caenorhabditis elegans, has a six-member gene family encoding vitellogenins, the yolk protein precursors. These genes are expressed exclusively in the intestine of the adult hermaphrodite. Here we report the cloning of all five members of the homologous gene family from anotherCaenorhabditis species,Caenorhabditis briggsae. Nucleotide sequence analysis of these genes reveals they are about 85% identical to theC. elegans genes in the coding regions. Oveerall similarity is much reduced in noncoding and flanking regions. However, two repeated heptamers, previously identified in the upstream regions of theC. elegans genes, are largely conserved in both location and sequence inC. briggsae. Conservation of certain of these heptamers suggests that proteins bound at these positions may be especially important to promoter function and/or regulation. Comparative sequence analysis also suggests the possibility that the first 70 bases of the vitellogenin mRNAs can be folded into stable secondary structures. Almost all base differences between the two species occur in sequences predicted to be unpaired, suggesting that the ability to form intrastrand base pairs has been selected duringCaenorhabditis evolution.     

A Genome-Wide Hybrid Incompatibility Landscape between Caenorhabditis briggsae and C. nigoni

Systematic characterization of ẖybrid incompatibility (HI) between related species remains the key to understanding speciation. The genetic basis of HI has been intensively studied in Drosophila species, but remains largely unknown in other species, including nematodes, which is mainly due to the lack of a sister species with which C. elegans can mate and produce viable progeny. The recent discovery of a C. briggsae sister species, C. nigoni, has opened up the possibility of dissecting the genetic basis of HI in nematode species. However, the paucity of dominant and visible marker prevents the efficient mapping of HI loci between the two species. To elucidate the genetic basis of speciation in nematode species, we first generated 96 chromosomally integrated GFP markers in the C. briggsae genome and mapped them into the defined locations by PCR and Next-Generation Sequencing (NGS). Aided by the marker, we backcrossed the GFP-associated C. briggsae genomic fragments into C. nigoni for at least 15 generations and produced 111 independent introgressions. The introgression fragments cover most of the C. briggsae genome. We finally dissected the patterns of HI by scoring the embryonic lethality, larval arrest, sex ratio and male sterility for each introgression line, through which we identified pervasive HI loci and produced a genome-wide landscape of HI between the two nematode species, the first of its type for any non-Drosophila species. The HI data not only provided insights into the genetic basis of speciation, but also established a framework for the possible cloning of HI loci between the two nematode species. Furthermore, the data on hybrids confirmed Haldane’s rule and suggested the presence of a large X effect in terms of fertility between the two species. Importantly, this work opens a new avenue for studying speciation genetics between nematode species and allows parallel comparison of the HI with that in Drosophila and other species.     

Comparative functional characterization of the CSR-1 22G-RNA pathway in Caenorhabditis nematodes

As a champion of small RNA research for two decades, Caenorhabditis elegans has revealed the essential Argonaute CSR-1 to play key nuclear roles in modulating chromatin, chromosome segregation and germline gene expression via 22G-small RNAs. Despite CSR-1 being preserved among diverse nematodes, the conservation and divergence in function of the targets of small RNA pathways remains poorly resolved. Here we apply comparative functional genomic analysis between C. elegans and Caenorhabditis briggsae to characterize the CSR-1 pathway, its targets and their evolution. C. briggsae CSR-1-associated small RNAs that we identified by immunoprecipitation-small RNA sequencing overlap with 22G-RNAs depleted in cbr-csr-1 RNAi-treated worms. By comparing 22G-RNAs and target genes between species, we defined a set of CSR-1 target genes with conserved germline expression, enrichment in operons and more slowly evolving coding sequences than other genes, along with a small group of evolutionarily labile targets. We demonstrate that the association of CSR-1 with chromatin is preserved, and show that depletion of cbr-csr-1 leads to chromosome segregation defects and embryonic lethality. This first comparative characterization of a small RNA pathway in Caenorhabditis establishes a conserved nuclear role for CSR-1 and highlights its key role in germline gene regulation across multiple animal species.     

Several Grassland Soil Nematode Species Are Insensitive to RNA-Mediated Interference

Phenotypic analysis of defects caused by RNA mediated interference (RNAi) in Caenorhabditis elegans has proven to be a powerful tool for determining gene function. In this study we investigated the effectiveness of RNAi in four non-model grassland soil nematodes, Oscheius sp FVV-2., Rhabditis sp, Mesorhabditis sp., and Acrobeloides sp. In contrast to reference experiments performed using C. elegans and Caenorhabditis briggsae, feeding bacteria expressing dsRNA and injecting dsRNA into the gonad did not produce the expected RNAi knockdown phenotypes in any of the grassland nematodes. Quantitative reverse-transcribed PCR (qRT-PCR) assays did not detect a statistically significant reduction in the mRNA levels of endogenous genes targeted by RNAi in Oscheius sp., and Mesorhabditis sp. From these studies we conclude that due to low effectiveness and inconsistent reproducibility, RNAi knockdown phenotypes in non-Caenorhabditis nematodes should be interpreted cautiously.     

Caenorhabditis briggsae and C. elegans: partial characterization of cuticle surface carbohydrates.

The presence of galactose, glucose, mannose, and N-acetylglucosamine on the exposed surface of the nematodes Caenorhabditis briggsae and C. elegans was indicated by specific binding of three iodinated plant lectins. Proteolysis experiments suggested the absence of digestible glycoproteins on the exposed surfaces of the two nematode species. High resolution micrographs of cuticle surface preparations labeled with cationized ferritin indicated that the negative charge-bearing molecules are more densely packed on the nematode surface than on animal plasma membranes.     

Molecular population genetics and phenotypic sensitivity to ethanol for a globally diverse sample of the nematode Caenorhabditis briggsae

New genomic resources and genetic tools of the past few years have advanced the nematode genus Caenorhabditis as a model for comparative biology. However, understanding of natural genetic variation at molecular and phenotypic levels remains rudimentary for most species in this genus, and for C. briggsae in particular. Here we characterize phenotypic variation in C. briggsae’s sensitivity to the potentially important and variable environmental toxin, ethanol, for globally diverse strains. We also quantify nucleotide variation in a new sample of 32 strains from four continents, including small islands, and for the closest‐known relative of this species (C. sp. 9). We demonstrate that C. briggsae exhibits little heritable variation for the effects of ethanol on the norm of reaction for survival and reproduction. Moreover, C. briggsae does not differ significantly from C. elegans in our assays of its response to this substance that both species likely encounter regularly in habitats of rotting fruit and vegetation. However, we uncover drastically more molecular genetic variation than was known previously for this species, despite most strains, including all island strains, conforming to the broad biogeographic patterns described previously. Using patterns of sequence divergence between populations and between species, we estimate that the self‐fertilizing mode of reproduction by hermaphrodites in C. briggsae likely evolved sometime between 0.9 and 10 million generations ago. These insights into C. briggsae’s natural history and natural genetic variation greatly expand the potential of this organism as an emerging model for studies in molecular and quantitative genetics, the evolution of development, and ecological genetics.     

Hydrolysis of aromatic β-glucosides by non-pathogenic bacteria confers a chemical weapon against predators

Bacteria present in natural environments such as soil have evolved multiple strategies to escape predation. We report that natural isolates of Enterobacteriaceae that actively hydrolyze plant-derived aromatic β-glucosides such as salicin, arbutin and esculin, are able to avoid predation by the bacteriovorous amoeba Dictyostelium discoideum and nematodes of multiple genera belonging to the family Rhabditidae. This advantage can be observed under laboratory culture conditions as well as in the soil environment. The aglycone moiety released by the hydrolysis of β-glucosides is toxic to predators and acts via the dopaminergic receptor Dop-1 in the case of Caenorhabditis elegans. While soil isolates of nematodes belonging to the family Rhabditidae are repelled by the aglycone, laboratory strains and natural isolates of Caenorhabditis sp. are attracted to the compound, mediated by receptors that are independent of Dop-1, leading to their death. The β-glucosides–positive (Bgl⁺) bacteria that are otherwise non-pathogenic can obtain additional nutrients from the dead predators, thereby switching their role from prey to predator. This study also offers an evolutionary explanation for the retention by bacteria of ‘cryptic’ or ‘silent’ genetic systems such as the bgl operon.     

A comparative study of sperm morphology, cytology and activation in Caenorhabditis elegans, Caenorhabditis remanei and Caenorhabditis briggsae

Studies of sterile mutants in Caenorhabditis elegans have uncovered new insights into fundamental aspects of gamete cell biology, development, and function at fertilization. The genome sequences of C. elegans, Caenorhabditis briggsae and Caenorhabditis remanei allow for informative comparative studies among these three species. Towards that end, we have examined wild-type sperm morphology and activation (spermiogenesis) in each. Light and electron microscopy studies reveal that general sperm morphology, organization, and ultrastructure are similar in all three species, and activation techniques developed for C. elegans were found to work well in both C. briggsae and C. remanei. Despite important differences in the reproductive mode between C. remanei and the other two species, most genes required for spermiogenesis are conserved in all three. Finally, we have also examined the subcellular distribution of sperm epitopes in C. briggsae and C. remanei that cross-react with anti-sera directed against C. elegans sperm proteins. The baseline data in this study will prove useful for the future analysis and interpretation of sperm gene function across nematode species.     

Functional constraint and divergence in the G protein family in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> and <Emphasis Type="Italic">Caenorhabditis briggsae</Emphasis>

Part of the challenge of the post-genomic world is to identify functional elements within the wide array of information generated by genome sequencing. Although cross-species comparisons and investigation of rates of sequence divergence are an efficient approach, the relationship between sequence divergence and functional conservation is not clear. Here, we use a comparative approach to examine questions of evolutionary rates and conserved function within the guanine nucleotide-binding protein (G protein) gene family in nematodes of the genus Caenorhabditis. In particular, we show that, in cases where the Caenorhabditis elegans ortholog shows a loss-of-function phenotype, G protein genes of C. elegans and Caenorhabditis briggsae diverge on average three times more slowly than G protein genes that do not exhibit any phenotype when mutated in C. elegans, suggesting that genes with loss of function phenotypes are subject to stronger selective constraints in relation to their function in both species. Our results also indicate that selection is as strong on G proteins involved in environmental perception as it is on those controlling other important processes. Finally, using phylogenetic footprinting, we identify a conserved non-coding motif present in multiple copies in the genomes of four species of Caenorhabditis. The presence of this motif in the same intron in the gpa-1 genes of C. elegans, C. briggsae and Caenorhabditis remanei suggests that it plays a role in the regulation of gpa-1, as well as other loci.Electronic Supplementary Material Supplementary material is available for this article at     

Morphological and Molecular Characterization of Phasmarhabditis huizhouensis sp. nov. (Nematoda: Rhabditidae), a New Rhabditid Nematode from South China

The genus Phasmarhabditis is an economically important group of rhabditid nematodes, to which the well-known slug-parasite P. hermaphrodita belongs. Despite the commercial use of Phasmarhabditis species as an attractive and promising approach for pest control, the taxonomy and systematics of this group of rhabditids are poorly understood, largely because of the lack of diagnostic morphological features and DNA sequences for distinguishing species or inferring phylogenetic relationship. During a nematode sampling effort for identifying free-living relatives of Caenorhabditis elegans in Huizhou City, Guangdong, China, a novel species belonging to the genus Phasmarhabditis was isolated from rotting leaves. Detailed morphology of the gonochoristic P. huizhouensis sp. nov. was described and illustrated. The adult female has a robust body, a relatively short and wide buccal capsule conjoined by a rhabditiform pharynx. Females are characterized by a short cupola-shaped tail end bearing a slender pointed tip, with the junction flanked by a pair of ‘rod-like’ phasmids. Males have an open peloderan bursa that is supported by 9 pairs of genital papillae and 1 terminal pair of phasmids. P. huizhouensis sp. nov. is morphologically very similar to the type species Phasmarhabditis papillosa but is distinguishable by its male caudal traits. The new species is readily differentiated from other taxa in the genus by its female tail shape. Molecular phylogenetic inferences based on small subunit (SSU) and the D2-D3 domain of large subunit (LSU) ribosomal DNA genes reveal that P. huizhouensis sp. nov. forms a unique branch in both phylogenies which is genetically related to P. hermaphrodita and other parasites such as Angiostoma spp. The host associations of P. huizhouensis sp. nov. and its ability to parasitize slugs are unknown.     

Evolution of dnmt-2 and mbd-2-like genes in the free-living nematodes Pristionchus pacificus, Caenorhabditis elegans and Caenorhabditis briggsae

Whole genome sequencing of several metazoan model organisms provides a platform for studying genome evolution. How representative are the genomes of these model organisms for their respective phyla? Within nematodes, for example, the free-living soil nematode Caenorhabditis elegans is a highly derived species with unusual genomic characters, such as a reduced Hox cluster (Curr. Biol., 13, 37–40) and the absence of a Hedgehog signaling system. Here, we describe the recent loss of a DNA methyltransferase-2 gene (dnmt-2) in C.elegans. A dnmt-2-like gene is present in the satellite model organism Pristionchus pacificus, another free-living nematode that diverged from C.elegans 200–300 million years ago. In contrast, C.elegans, Caenorhabditis briggsae and P.pacificus all contain an mbd-2-like gene, which encodes another essential component of the methylation system of higher animals and fungi. Cel-mbd-2 is expressed throughout development and RNA interference (RNAi) experiments result in variable phenotypes. In contrast, Cbr-mbd-2 RNAi results in paralyzed larval or adult worms suggesting recent changes of gene function within the genus Caenorhabditis. We speculate that both genes were part of an ancestral DNA methylation system in nematodes and that gene loss and sequence divergence have abolished DNA methylation in C.elegans.     

Nematode selenoproteome: the use of the selenocysteine insertion system to decode one codon in an animal genome?

Selenocysteine (Sec) is co-translationally inserted into selenoproteins in response to codon UGA with the help of the selenocysteine insertion sequence (SECIS) element. The number of selenoproteins in animals varies, with humans having 25 and mice having 24 selenoproteins. To date, however, only one selenoprotein, thioredoxin reductase, has been detected in Caenorhabditis elegans, and this enzyme contains only one Sec. Here, we characterize the selenoproteomes of C.elegans and Caenorhabditis briggsae with three independent algorithms, one searching for pairs of homologous nematode SECIS elements, another searching for Cys- or Sec-containing homologs of potential nematode selenoprotein genes and the third identifying Sec-containing homologs of annotated nematode proteins. These methods suggest that thioredoxin reductase is the only Sec-containing protein in the C.elegans and C.briggsae genomes. In contrast, we identified additional selenoproteins in other nematodes. Assuming that Sec insertion mechanisms are conserved between nematodes and other eukaryotes, the data suggest that nematode selenoproteomes were reduced during evolution, and that in an extreme reduction case Sec insertion systems probably decode only a single UGA codon in C.elegans and C.briggsae genomes. In addition, all detected genes had a rare form of SECIS element containing a guanosine in place of a conserved adenosine present in most other SECIS structures, suggesting that in organisms with small selenoproteomes SECIS elements may change rapidly.     

Effects of Infection by Belonolaimus longicaudatus on Rooting Dynamics among St. Augustinegrass and Bermudagrass Genotypes

Understanding rooting dynamics using the minirhizotron technique is useful for cultivar selection and to quantify nematode damage to roots. A 2-yr microplot study including five bermudagrass (‘Tifway’, Belonolaimus longicaudatus susceptible; two commercial cultivars [TifSport and Celebration] and two genotypes [‘BA132’ and ‘PI 291590’], which have been reported to be tolerant to B. longicaudatus) and two St. Augustinegrass (‘FX 313’, susceptible, and ‘Floratam’ that was reported as tolerant to B. longicaudatus) genotypes in a 5 x 2 and 2 x 2 factorial design with four replications, respectively, was initiated in 2012. Two treatments included were uninoculated and B. longicaudatus inoculated. In situ root images were captured each month using a minirhizotron camera system from April to September of 2013 and 2014. Mixed models analysis and comparison of least squares means indicated significant differences in root parameters studied across the genotypes and soil depths of both grass species. ‘Celebration’, ‘TifSport’ and ‘PI 291590’ bermudagrass, and ‘Floratam’ St. Augustinegrass had significantly different root parameters compared to the corresponding susceptible genotypes (P ≤ 0.05). Only ‘TifSport’ had no significant root loss when infested with B. longicaudatus compared to non-infested. ‘Celebration’ and ‘PI 291590’ had significant root loss but retained significantly greater root densities than ‘Tifway’ in B. longicaudatus-infested conditions (P ≤ 0.05). Root lengths were greater at the 0 to 5 cm depth followed by 5 to 10 and 10 to 15 cm of vertical soil depth for both grass species (P ≤ 0.05). ‘Celebration’, ‘TifSport’, and ‘PI 291590’ had better root vigor against B. longicaudatus compared to Tifway.     

Multi-Environment Model Estimation for Motility Analysis of Caenorhabditis elegans

The nematode Caenorhabditis elegans is a well-known model organism used to investigate fundamental questions in biology. Motility assays of this small roundworm are designed to study the relationships between genes and behavior. Commonly, motility analysis is used to classify nematode movements and characterize them quantitatively. Over the past years, C. elegans'' motility has been studied across a wide range of environments, including crawling on substrates, swimming in fluids, and locomoting through microfluidic substrates. However, each environment often requires customized image processing tools relying on heuristic parameter tuning. In the present study, we propose a novel Multi-Environment Model Estimation (MEME) framework for automated image segmentation that is versatile across various environments. The MEME platform is constructed around the concept of Mixture of Gaussian (MOG) models, where statistical models for both the background environment and the nematode appearance are explicitly learned and used to accurately segment a target nematode. Our method is designed to simplify the burden often imposed on users; here, only a single image which includes a nematode in its environment must be provided for model learning. In addition, our platform enables the extraction of nematode ‘skeletons’ for straightforward motility quantification. We test our algorithm on various locomotive environments and compare performances with an intensity-based thresholding method. Overall, MEME outperforms the threshold-based approach for the overwhelming majority of cases examined. Ultimately, MEME provides researchers with an attractive platform for C. elegans'' segmentation and ‘skeletonizing’ across a wide range of motility assays.