Caenorhabditis Briggsae Research Articles

Caenorhabditis microbiota: worm guts get populated.

Until recently, almost nothing has been known about the natural microbiota of the model nematode Caenorhabditis elegans. Reporting their research in BMC Biology, Dirksen and colleagues describe the first sequencing effort to characterize the gut microbiota of environmentally isolated C. elegans and the related taxa Caenorhabditis briggsae and Caenorhabditis remanei In contrast to the monoxenic, microbiota-free cultures that are studied in hundreds of laboratories, it appears that natural populations of Caenorhabditis harbor distinct microbiotas.

Evolutionary expansion of nematode-specific glycine-rich secreted peptides

Genome-wide comparisons across 10 species from algae Guillardia theta to mammal human indicated that Caenorhabditis elegans and Caenorhabditis briggsae were highly enriched for glycine-rich secreted peptides (GRSPs) (110 GRSPs in C. elegans and 93 in C. briggsae) in this study. Chromosomal mapping showed that most GRSPs were clustered on the two nematode genomes [103 (93.64%) in C. elegans and 82 (88.17%) in C. briggsae], which could be divided into 18 cluster units in C. elegans and 13 in C. briggsae, respectively. Except for four C. elegans GRSPs clusters without matching clusters in C. briggsae, all other GRSPs clusters had paired synteny block between the two nematode genomes. Analyzing transcriptome datasets quantified by microarray indicated extensive genome-wide co-expression of GRSPs clusters after C. elegans infections. Highly homologous coding sequences and conserved exon–intron structures indicated that GRSPs tight clusters were likely derived from local DNA duplications. Phylogenetic conservation of synteny blocks between their genomes, co-expression of GRSPs clusters after C. elegans infections, and strong purifying selections of coding sequences may indicate evolutionary constraints acting on C. elegans to guarantee that C. elegans could mount rapid systematic responses to infections by co-expression, co-regulation, and co-functionality of GRSPs clusters.

Conservation of anatomically restricted glycosaminoglycan structures in divergent nematode species.

Heparan sulfates (HS) are glycosaminoglycans of the extracellular matrices and characterized by complex modification patterns owing to sulfations, epimerization, and acetylation. Distinct HS modification patterns have been shown to modulate protein-protein interactions during development in general and of the nervous system in particular. This has led to the heparan sulfate code hypothesis, which posits that specifically modified HS epitopes are distributed in a tissue and cell-specific fashion to orchestrate neural circuit formation. Whether an HS code exists in vivo, how specific or how evolutionarily conserved the anatomical distribution of an HS code may be has remained unknown. Here we conduct a systematic comparison of HS modification patterns in the nematode Caenorhabditis elegans using transgenic expression of 33 different HS-specific single chain variable fragment antibodies. We find that some HS modification patterns are widely distributed in the nervous system. In contrast, other HS modification patterns appear highly cell-specific in both non-neuronal and neuronal cells. Some patterns can be as restricted in their localization as to single neurites or synaptic connections between two neurons. This restricted anatomical localization of specific HS patterns can be evolutionarily conserved over a span of 80-100 million years in the divergent nematode species Caenorhabditis briggsae suggesting structural and, possibly functional conservation of glycosaminoglycan structures similar to proteins. These findings suggest a HS code with subcellularly localized, unique glycan identities in the nervous system.

Suppression of F1 Male-Specific Lethality in Caenorhabditis Hybrids by cbr-him-8.

Haldane’s Rule and Darwin’s Corollary to Haldane’s Rule are the observations that heterogametic F1 hybrids are frequently less fit than their homogametic siblings, and that asymmetric results are often obtained from reciprocal hybrid crosses. In Caenorhabditis, Haldane’s Rule and Darwin’s Corollary have been observed in several hybrid crosses, including crosses of Caenorhabditis briggsae and C. nigoni. Fertile F1 females are obtained from reciprocal crosses. However, F1 males obtained from C. nigoni mothers are sterile and F1 males obtained from C. briggsae die during embryogenesis. We have identified cbr-him-8 as a recessive maternal-effect suppressor of F1 hybrid male-specific lethality in this combination of species. This result implicates epigenetic meiotic silencing in the suppression of F1 male-specific lethality. It is also shown that F1 males bearing a C. briggsae X chromosome are fertile. When crossed to C. briggsae hermaphrodites or F1 females derived from C. briggsae hermaphrodites, viable F2 and backcross (B2) progeny were obtained. Sibling males that possessed a C. nigoni X chromosome were sterile. Therefore, the sterility of F1 males bearing a C. nigoni X chromosome must result from dysgenic interactions between the X chromosome of C. nigoni and the autosomes of C. briggsae. The fertility of F1 males bearing a C. briggsae X chromosome provides an opportunity to identify C. nigoni loci that prevent spermatogenesis, and hence hermaphroditic reproduction, in diplo-X hybrids.

Selective MS screening reveals a sex pheromone in Caenorhabditis briggsae and species-specificity in indole ascaroside signalling.

The indole ascarosides (icas) represent a highly potent class of nematode-derived modular signalling components that integrate structural inputs from amino acid, carbohydrate, and fatty acid metabolism. Comparative analysis of the crude exo-metabolome of hermaphroditic Caenorhabditis briggsae using a highly sensitive mass spectrometric screen reveals an indole ascaroside blend dominated by two new components. The structures of isolated icas#2 and icas#6.2 were determined by NMR spectroscopy and confirmed by total synthesis and chemical correlation. Low atto- to femtomolar amounts of icas#2 and icas#6.2 act in synergism to attract males indicating a function as sex pheromone. Comparative analysis of 14 Caenorhabditis species further demonstrates that species-specific indole ascaroside biosynthesis is highly conserved in the Elegans group. Functional characterization of the dominating indole ascarosides icas#2, icas#3, and icas#9 reveals a high degree of species-specificity and considerable variability with respect to gender-specificity, thus, confirming that indole ascarosides modulate different biological functions within the Elegans group. Although the nematode response was usually most pronounced towards conspecific signals, Caenorhabditis brenneri, the only species of the Elegans group that does not produce any indole ascarosides, exhibits a robust response to icas#2 suggesting the potential for interspecies interactions.

Mitochondrial-Nuclear Epistasis Impacts Fitness and Mitochondrial Physiology of Interpopulation Caenorhabditis briggsae Hybrids.

In order to identify the earliest genetic changes that precipitate species formation, it is useful to study genetic incompatibilities that cause only mild dysfunction when incompatible alleles are combined in an interpopulation hybrid. Such hybridization within the nematode species Caenorhabditis briggsae has been suggested to result in selection against certain combinations of nuclear and mitochondrial alleles, raising the possibility that mitochondrial–nuclear (mitonuclear) epistasis reduces hybrid fitness. To test this hypothesis, cytoplasmic–nuclear hybrids (cybrids) were created to purposefully disrupt any epistatic interactions. Experimental analysis of the cybrids suggests that mitonuclear discord can result in decreased fecundity, increased lipid content, and increased mitochondrial reactive oxygen species levels. Many of these effects were asymmetric with respect to cross direction, as expected if cytoplasmic–nuclear Dobzhansky-Muller incompatibilities exist. One such effect is consistent with the interpretation that disrupting coevolved mitochondrial and nuclear loci impacts mitochondrial function and organismal fitness. These findings enhance efforts to study the genesis, identity, and maintenance of genetic incompatibilities that precipitate the speciation process.

Quantitative trait loci mapping of dauer larvae development in growing populations of Caenorhabditis briggsae

The life cycles of many free-living nematodes contain developmental switches that allow individuals to either develop directly to adulthood, or to arrest development as a stress resistant and long-lived dauer larval stage. Here, in a panel of Caenorhabditis briggsae recombinant inbred lines derived from the isolates HK104 x AF16, we use methodologies developed for C.elegans to map quantitative trait loci (QTLs) affecting the number of dauer larvae present at the point of food patch exhaustion. These analyses provide strong support for three QTLs and are suggestive of a further two.

Genome sequence and comparative analysis of a putative entomopathogenic Serratia isolated from Caenorhabditis briggsae.

BackgroundEntomopathogenic associations between nematodes in the genera Steinernema and Heterorhabdus with their cognate bacteria from the bacterial genera Xenorhabdus and Photorhabdus, respectively, are extensively studied for their potential as biological control agents against invasive insect species. These two highly coevolved associations were results of convergent evolution. Given the natural abundance of bacteria, nematodes and insects, it is surprising that only these two associations with no intermediate forms are widely studied in the entomopathogenic context. Discovering analogous systems involving novel bacterial and nematode species would shed light on the evolutionary processes involved in the transition from free living organisms to obligatory partners in entomopathogenicity.ResultsWe report the complete genome sequence of a new member of the enterobacterial genus Serratia that forms a putative entomopathogenic complex with Caenorhabditis briggsae. Analysis of the 5.04 MB chromosomal genome predicts 4599 protein coding genes, seven sets of ribosomal RNA genes, 84 tRNA genes and a 64.8 KB plasmid encoding 74 genes. Comparative genomic analysis with three of the previously sequenced Serratia species, S. marcescens DB11 and S. proteamaculans 568, and Serratia sp. AS12, revealed that these four representatives of the genus share a core set of ~3100 genes and extensive structural conservation. The newly identified species shares a more recent common ancestor with S. marcescens with 99 % sequence identity in rDNA sequence and orthology across 85.6 % of predicted genes. Of the 39 genes/operons implicated in the virulence, symbiosis, recolonization, immune evasion and bioconversion, 21 (53.8 %) were present in Serratia while 33 (84.6 %) and 35 (89 %) were present in Xenorhabdus and Photorhabdus EPN bacteria respectively.ConclusionThe majority of unique sequences in Serratia sp. SCBI (South African Caenorhabditis briggsae Isolate) are found in ~29 genomic islands of 5 to 65 genes and are enriched in putative functions that are biologically relevant to an entomopathogenic lifestyle, including non-ribosomal peptide synthetases, bacteriocins, fimbrial biogenesis, ushering proteins, toxins, secondary metabolite secretion and multiple drug resistance/efflux systems. By revealing the early stages of adaptation to this lifestyle, the Serratia sp. SCBI genome underscores the fact that in EPN formation the composite end result – killing, bioconversion, cadaver protection and recolonization- can be achieved by dissimilar mechanisms. This genome sequence will enable further study of the evolution of entomopathogenic nematode-bacteria complexes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1697-8) contains supplementary material, which is available to authorized users.

Gametic selection, developmental trajectories, and extrinsic heterogeneity in Haldane's rule.

Deciphering the genetic and developmental causes of the disproportionate rarity, inviability, and sterility of hybrid males, Haldane's rule, is important for understanding the evolution of reproductive isolation between species. Moreover, extrinsic and prezygotic factors can contribute to the magnitude of intrinsic isolation experienced between species with partial reproductive compatibility. Here, we use the nematodes Caenorhabditis briggsae and C. nigoni to quantify the sensitivity of hybrid male viability to extrinsic temperature and developmental timing, and test for a role of mito-nuclear incompatibility as a genetic cause. We demonstrate that hybrid male inviability manifests almost entirely as embryonic, not larval, arrest and is maximal at the lowest rearing temperatures, indicating an intrinsic-by-extrinsic interaction to hybrid inviability. Crosses using mitochondrial substitution strains that have reciprocally introgressed mitochondrial and nuclear genomes show that mito-nuclear incompatibility is not a dominant contributor to postzygotic isolation and does not drive Haldane's rule in this system. Crosses also reveal that competitive superiority of X-bearing sperm provides a novel means by which postmating prezygotic factors exacerbate the rarity of hybrid males. These findings highlight the important roles of gametic, developmental, and extrinsic factors in modulating the manifestation of Haldane's rule.

Selfish Mitochondrial DNA Proliferates and Diversifies in Small, but not Large, Experimental Populations of Caenorhabditis briggsae.

Evolutionary interactions across levels of biological organization contribute to a variety of fundamental processes including genome evolution, reproductive mode transitions, species diversification, and extinction. Evolutionary theory predicts that so-called “selfish” genetic elements will proliferate when the host effective population size (Ne) is small, but direct tests of this prediction remain few. We analyzed the evolutionary dynamics of deletion-containing mitochondrial DNA (ΔmtDNA) molecules, previously characterized as selfish elements, in six different natural strains of the nematode Caenorhabditis briggsae allowed to undergo experimental evolution in a range of population sizes (N = 1, 10, 100, and 1,000) for a maximum of 50 generations. Mitochondrial DNA (mtDNA) was analyzed for replicate lineages at each five-generation time point. Ten different ΔmtDNA molecule types were observed and characterized across generations in the experimental populations. Consistent with predictions from evolutionary theory, lab lines evolved in small-population sizes (e.g., nematode N = 1) were more susceptible to accumulation of high levels of preexisting ΔmtDNA compared with those evolved in larger populations. New ΔmtDNA elements were observed to increase in frequency and persist across time points, but almost exclusively at small population sizes. In some cases, ΔmtDNA levels decreased across generations when population size was large (nematode N = 1,000). Different natural strains of C. briggsae varied in their susceptibilities to ΔmtDNA accumulation, owing in part to preexisting compensatory mtDNA alleles in some strains that prevent deletion formation. This analysis directly demonstrates that the evolutionary trajectories of ΔmtDNA elements depend upon the population-genetic environments and molecular-genetic features of their hosts.

Cellular hallmarks reveal restricted aerobic metabolism at thermal limits.

All organisms live within a given thermal range, but little is known about the mechanisms setting the limits of this range. We uncovered cellular features exhibiting signature changes at thermal limits in Caenorhabditis elegans embryos. These included changes in embryo size and shape, which were also observed in Caenorhabditis briggsae, indicating evolutionary conservation. We hypothesized that such changes could reflect restricted aerobic capacity at thermal limits. Accordingly, we uncovered that relative respiration in C. elegans embryos decreases at the thermal limits as compared to within the thermal range. Furthermore, by compromising components of the respiratory chain, we demonstrated that the reliance on aerobic metabolism is reduced at thermal limits. Moreover, embryos thus compromised exhibited signature changes in size and shape already within the thermal range. We conclude that restricted aerobic metabolism at the thermal limits contributes to setting the thermal range in a metazoan organism.

Draft genome of neurotropic nematode parasite Angiostrongylus cantonensis, causative agent of human eosinophilic meningitis

Angiostrongylus cantonensis is a bursate nematode parasite that causes eosinophilic meningitis (or meningoencephalitis) in humans in many parts of the world. The genomic data from A. cantonensis will form a useful resource for comparative genomic and chemogenomic studies to aid the development of diagnostics and therapeutics. We have sequenced, assembled and annotated the genome of A. cantonensis. The genome size is estimated to be ∼260Mb, with 17,280 genomic scaffolds, 91X coverage, 81.45% for complete and 93.95% for partial score based on CEGMA analysis of genome completeness. The number of predicted genes of ≥300bp was 17,482. A total of 7737 predicted protein-coding genes of ≥50 amino acids were identified in the assembled genome. Among the proteins of known function, kinases are the most abundant followed by transferases. The draft genome contains 34 excretory–secretory proteins (ES), a minimum of 44 Nematode Astacin (NAS) metalloproteases, 12 Homeobox (HOX) genes, and 30 neurotransmitters. The assembled genome size (260Mb) is larger than those of Pristionchus pacificus, Caenorhabditis elegans, Necator americanus, Caenorhabditis briggsae, Trichinella spiralis, Brugia malayi and Loa loa, but smaller than Haemonchus contortus and Ascaris suum. The repeat content (25%) is similar to H. contortus. The GC content (41.17%) is lower compared to P. pacificus (42.7%) and H. contortus (43.1%) but higher compared to C. briggsae (37.69%), A. suum (37.9%) and N. americanus (40.2%) while the scaffold N50 is 42,191. This draft genome will facilitate the understanding of many unresolved issues on the parasite and the disorder it causes.

Full-genome evolutionary histories of selfing, splitting, and selection in Caenorhabditis.

The nematode Caenorhabditis briggsae is a model for comparative developmental evolution with C. elegans. Worldwide collections of C. briggsae have implicated an intriguing history of divergence among genetic groups separated by latitude, or by restricted geography, that is being exploited to dissect the genetic basis to adaptive evolution and reproductive incompatibility; yet, the genomic scope and timing of population divergence is unclear. We performed high-coverage whole-genome sequencing of 37 wild isolates of the nematode C. briggsae and applied a pairwise sequentially Markovian coalescent (PSMC) model to 703 combinations of genomic haplotypes to draw inferences about population history, the genomic scope of natural selection, and to compare with 40 wild isolates of C. elegans. We estimate that a diaspora of at least six distinct C. briggsae lineages separated from one another approximately 200,000 generations ago, including the “Temperate” and “Tropical” phylogeographic groups that dominate most samples worldwide. Moreover, an ancient population split in its history approximately 2 million generations ago, coupled with only rare gene flow among lineage groups, validates this system as a model for incipient speciation. Low versus high recombination regions of the genome give distinct signatures of population size change through time, indicative of widespread effects of selection on highly linked portions of the genome owing to extreme inbreeding by self-fertilization. Analysis of functional mutations indicates that genomic context, owing to selection that acts on long linkage blocks, is a more important driver of population variation than are the functional attributes of the individually encoded genes.

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.

Mutations in Caenorhabditis briggsae identify new genes important for limiting the response to EGF signaling during vulval development.

Studies of vulval development in the nematode C. elegans have identified many genes that are involved in cell division and differentiation processes. Some of these encode components of conserved signal transduction pathways mediated by EGF, Notch, and Wnt. To understand how developmental mechanisms change during evolution, we are doing a comparative analysis of vulva formation in C. briggsae, a species that is closely related to C. elegans. Here, we report 14 mutations in 7 Multivulva (Muv) genes in C. briggsae that inhibit inappropriate division of vulval precursors. We have developed a new efficient and cost-effective gene mapping method to localize Muv mutations to small genetic intervals on chromosomes, thus facilitating cloning and functional studies. We demonstrate the utility of our method by determining molecular identities of three of the Muv genes that include orthologs of Cel-lin-1 (ETS) and Cel-lin-31 (Winged-Helix) of the EGF-Ras pathway and Cel-pry-1 (Axin), of the Wnt pathway. The remaining four genes reside in regions that lack orthologs of known C. elegans Muv genes. Inhibitor studies demonstrate that the Muv phenotype of all four new genes is dependent on the activity of the EGF pathway kinase, MEK. One of these, Cbr-lin(gu167), shows modest increase in the expression of Cbr-lin-3/EGF compared to wild type. These results argue that while Cbr-lin(gu167) may act upstream of Cbr-lin-3/EGF, the other three genes influence the EGF pathway downstream or in parallel to Cbr-lin-3. Overall, our findings demonstrate that the genetic program underlying a conserved developmental process includes both conserved and divergent functional contributions.

Molecular characterization of protease activity in Serratia sp. strain SCBI and its importance in cytotoxicity and virulence.

A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex.

Dependence of the sperm/oocyte decision on the nucleosome remodeling factor complex was acquired during recent Caenorhabditis briggsae evolution.

The major families of chromatin remodelers have been conserved throughout eukaryotic evolution. Because they play broad, pleiotropic roles in gene regulation, it was not known if their functions could change rapidly. Here, we show that major alterations in the use of chromatin remodelers are possible, because the nucleosome remodeling factor (NURF) complex has acquired a unique role in the sperm/oocyte decision of the nematode Caenorhabditis briggsae. First, lowering the activity of C. briggsae NURF-1 or ISW-1, the core components of the NURF complex, causes germ cells to become oocytes rather than sperm. This observation is based on the analysis of weak alleles and null mutations that were induced with TALENs and on RNA interference. Second, qRT–polymerase chain reaction data show that the C. briggsae NURF complex promotes the expression of Cbr-fog-1 and Cbr-fog-3, two genes that control the sperm/oocyte decision. This regulation occurs in the third larval stage and affects the expression of later spermatogenesis genes. Third, double mutants reveal that the NURF complex and the transcription factor TRA-1 act independently on Cbr-fog-1 and Cbr-fog-3. TRA-1 binds both promoters, and computer analyses predict that these binding sites are buried in nucleosomes, so we suggest that the NURF complex alters chromatin structure to allow TRA-1 access to Cbr-fog-1 and Cbr-fog-3. Finally, lowering NURF activity by mutation or RNA interference does not affect this trait in other nematodes, including the sister species C. nigoni, so it must have evolved recently. We conclude that altered chromatin remodeling could play an important role in evolutionary change.

Worm peptidomics

Bioactive peptides are present in all metazoan species where they orchestrate diverse functions. In the last decade, high-throughput approaches based on mass spectrometry helped the identification of endogenously occurring peptides in different species. We here review biochemical strategies to obtain sequence information of natural (non-tryptic) peptides in Caenorhabditis elegans and in the related nematodes Caenorhabditis briggsae and Ascaris suum with particular attention for sample preparation and methodology. In addition, we describe seven new C. elegans neuropeptides that we recently discovered by sequencing additional peptides. Finally, we explain how differential peptidomics approaches were used to characterize key neuropeptide processing enzymes.

Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon.

We have generated a recombinant Mos1 transposon that can insert up to 45 kb transgenes into the C. elegans genome. The minimal Mos1 transposon (miniMos) is 550 bp long and inserts DNA into the genome at high frequency (~60% of injected animals). Genetic and antibiotic markers can be used for selection and the transposon is active in C. elegans isolates and C. briggsae. We have used the miniMos transposon to generate six universal MosSCI landing sites that allow targeted transgene insertion with a single targeting vector into permissive expression sites on all autosomes. We have also generated two collections of strains: A set of bright fluorescent insertions that are useful as dominant, genetic balancers and a set of lacO insertions to track genome position.

Identification of C. elegans & C. briggsae miRNAs by modified miRsearch

In this study, a modified miRsearch program was developed in C++ for the detection of miRNAs. All the mature miRNA sequences of Caenorhabditis elegans, Caenorhabditis briggsae, Caenorhabditis remanei, Drosophila melanogaster, Homo sapiens and Rattus norvegicus available in miRbase was searched by this program for homologous sequences with a maximum of 3-mismatches in the chromosomes of C. elegans excluding the miRNAs of C. elegans. The same strategy was repeated for C. briggsae excluding C. briggsae miRNAs. The probable pre-miRNA sequences with stem loop secondary structures were assessed by implementation of longest-common-subsequence (LCS) algorithm with appropriate scoring system. As miRNA genes could be on either strand, each sequence was searched in both forward and reverse strands. The putative miRNAs were viewed through Mapviewer to identify their intronic or intergenic location in C. elegans and C. briggsae genomes. Further, the quality of stem-loop formation of the remaining pre-miRNA sequences was assessed through RNAFOLD. This algorithm will be helpful in detection of potential miRNAs in future sequencing data, making this an invaluable tool for miRNA prediction.