Glial expression of a steroidogenic enzyme underlies natural variation in hitchhiking behavior.

Phoresy is an interspecies interaction that facilitates spatial dispersal by attaching to a more mobile species. Hitchhiking species have evolved specific traits for physical contact and successful phoresy, but the regulatory mechanisms involved in such traits and their evolution are largely unexplored. The nematode Caenorhabditis elegans displays a hitchhiking behavior known as nictation during its stress-induced developmental stage. Dauer-specific nictation behavior has an important role in natural C. elegans populations, which experience boom-and-bust population dynamics. In this study, we investigated the nictation behavior of 137 wild C. elegans strains sampled throughout the world. We identified species-wide natural variation in nictation and performed a genome-wide association mapping. We show that the variants in the promoter of nta-1, encoding a putative steroidogenic enzyme, underlie differences in nictation. This difference is due to the changes in nta-1 expression in glial cells, which implies that glial steroid metabolism regulates phoretic behavior. Population genetic analysis and geographic distribution patterns suggest that balancing selection maintained two nta-1 haplotypes that existed in ancestral C. elegans populations. Our findings contribute to further understanding of the molecular mechanism of species interaction and the maintenance of genetic diversity within natural populations.

Cyprocide selectively kills nematodes via cytochrome P450 bioactivation.

Left unchecked, plant-parasitic nematodes have the potential to devastate crops globally. Highly effective but non-selective nematicides are justifiably being phased-out, leaving farmers with limited options for managing nematode infestation. Here, we report our discovery of a 1,3,4-oxadiazole thioether scaffold called Cyprocide that selectively kills nematodes including diverse species of plant-parasitic nematodes. Cyprocide is bioactivated into a lethal reactive electrophilic metabolite by specific nematode cytochrome P450 enzymes. Cyprocide fails to kill organisms beyond nematodes, suggesting that the targeted lethality of this pro-nematicide derives from P450 substrate selectivity. Our findings demonstrate that Cyprocide is a selective nematicidal scaffold with broad-spectrum activity that holds the potential to help safeguard our global food supply.

Genetic variants that modify neuroendocrine gene expression and foraging behavior of C. elegans.

The molecular mechanisms underlying diversity in animal behavior are not well understood. A major experimental challenge is determining the contribution of genetic variants that affect neuronal gene expression to differences in behavioral traits. In Caenorhabditis elegans, the neuroendocrine transforming growth factor-ß ligand, DAF-7, regulates diverse behavioral responses to bacterial food and pathogens. The dynamic neuron-specific expression of daf-7 is modulated by environmental and endogenous bacteria-derived cues. Here, we investigated natural variation in the expression of daf-7 from the ASJ pair of chemosensory neurons. We identified common genetic variants in gap-2, encoding a Ras guanosine triphosphatase (GTPase)-activating protein homologous to mammalian synaptic Ras GTPase-activating protein, which modify daf-7 expression cell nonautonomously and promote exploratory foraging behavior in a partially DAF-7-dependent manner. Our data connect natural variation in neuron-specific gene expression to differences in behavior and suggest that genetic variation in neuroendocrine signaling pathways mediating host-microbe interactions may give rise to diversity in animal behavior.

Natural variation in protein kinase D modifies alcohol sensitivity in Caenorhabditis elegans.

Differences in naïve alcohol sensitivity between individuals are a strong predictor of later life alcohol use disorders (AUD). However, the genetic bases for alcohol sensitivity (beyond ethanol metabolism) and pharmacological approaches to modulate alcohol sensitivity remain poorly understood. We used a high-throughput behavioral screen to measure acute behavioral sensitivity to alcohol, a model of intoxication, in a genetically diverse set of over 150 wild strains of the nematode Caenorhabditis elegans. We performed a genome-wide association study to identify loci that underlie natural variation in alcohol sensitivity. We identified five quantitative trait loci (QTL) and further show that variants in the C. elegans ortholog of protein kinase D, dkf-2, likely underlie the chromosome V QTL. We found that resistance to intoxication was conferred by dkf-2 loss-of-function mutations as well as partly by a PKD inhibitor in a dkf-2-dependent manner. Protein kinase D might represent a conserved, druggable target to modify alcohol sensitivity with application towards AUD.

Nav1.2 channel mutations preventing fast inactivation lead to SCN2A encephalopathy.

SCN2A gene-related early-infantile developmental and epileptic encephalopathy (EI-DEE) is a rare and severe disorder that manifests in early infancy. SCN2A mutations affecting the fast inactivation gating mechanism can result in altered voltage dependence and incomplete inactivation of the encoded neuronal Nav1.2 channel and lead to abnormal neuronal excitability. In this study, we evaluated clinical data of seven missense Nav1.2 variants associated with DEE and performed molecular dynamics simulations, patch-clamp electrophysiology, and dynamic clamp real-time neuronal modelling to elucidate the molecular and neuron-scale phenotypic consequences of the mutations. The N1662D mutation almost completely prevented fast inactivation without affecting activation. The comparison of wild-type and N1662D channel structures suggested that the ambifunctional hydrogen bond formation between residues N1662 and Q1494 is essential for fast inactivation. Fast inactivation could also be prevented with engineered Q1494A or Q1494L Nav1.2 channel variants, whereas Q1494E or Q1494 K variants resulted in incomplete inactivation and persistent current. Molecular dynamics simulations revealed a reduced affinity of the hydrophobic IFM-motif to its receptor site with N1662D and Q1494L variants relative to wild-type. These results demonstrate that the interactions between N1662 and Q1494 underpin the stability and the orientation of the inactivation gate and are essential for the development of fast inactivation. Six DEE-associated Nav1.2 variants, with mutations mapped to channel segments known to be implicated in fast inactivation were also evaluated. Remarkably, the L1657P variant also prevented fast inactivation and produced biophysical characteristics that were similar to those of N1662D, whereas the M1501 V, M1501T, F1651C, P1658S, and A1659 V variants resulted in biophysical properties that were consistent with gain-of-function and enhanced action potential firing of hybrid neurons in dynamic action potential clamp experiments. Paradoxically, low densities of N1662D or L1657P currents potentiated action potential firing, whereas increased densities resulted in sustained depolarization. Our results provide novel structural insights into the molecular mechanism of Nav1.2 channel fast inactivation and inform treatment strategies for SCN2A-related EI-DEE. The contribution of non-inactivating Nav1.2 channels to neuronal excitability may constitute a distinct cellular mechanism in the pathogenesis of SCN2A-related DEE.

Further delineation of short-chain enoyl-CoA hydratase deficiency in the Pacific population.

Short-chain enoyl-coA hydratase (SCEH) deficiency due to biallelic pathogenic ECHS1 variants was first reported in 2014 in association with Leigh syndrome (LS) and increased S-(2-carboxypropyl)cysteine excretion. It is potentially treatable with a valine-restricted, high-energy diet and emergency regimen. Recently, Simon et al. described four Samoan children harbouring a hypomorphic allele (c.489G > A, p.Pro163=) associated with reduced levels of normally-spliced mRNA. This synonymous variant, missed on standard genomic testing, is prevalent in the Samoan population (allele frequency 0.17). Patients with LS and one ECHS1 variant were identified in NZ and Australian genomic and clinical databases. ECHS1 sequence data were interrogated for the c.489G > A variant and clinical data were reviewed. Thirteen patients from 10 families were identified; all had Pacific ancestry including Samoan, Maori, Cook Island Maori, and Tokelauan. All developed bilateral globus pallidi lesions, excluding one pre-symptomatic infant. Symptom onset was in early childhood, and was triggered by illness or starvation in 9/13. Four of 13 had exercise-induced dyskinesia, 9/13 optic atrophy and 6/13 nystagmus. Urine S-(2-carboxypropyl)cysteine-carnitine and other SCEH-related metabolites were normal or mildly increased. Functional studies demonstrated skipping of exon four and markedly reduced ECHS1 protein. These data provide further support for the pathogenicity of this ECHS1 variant which is also prevalent in Maori, Cook Island Maori, and Tongan populations (allele frequency 0.14-0.24). It highlights the need to search for a second variant in apparent heterozygotes with an appropriate phenotype, and has implications for genetic counselling in family members who are heterozygous for the more severe ECHS1 alleles. SYNOPSIS: Short-chain enoyl-CoA hydratase deficiency is a frequent cause of Leigh-like disease in Maori and wider-Pacific populations, due to the high carrier frequency of a hypomorphic ECHS1 variant c.489G > A, p.[Pro163=, Phe139Valfs*65] that may be overlooked by standard genomic testing.

Gene duplication and evolutionary plasticity of lin-12/Notch gene function in Caenorhabditis.

Gene duplication is an important substrate for the evolution of new gene functions, but the impacts of gene duplicates on their own activities and on the developmental networks in which they act are poorly understood. Here, we use a natural experiment of lin-12/Notch gene duplication within the nematode genus Caenorhabditis, combined with characterization of loss- and gain-of-function mutations, to uncover functional distinctions between the duplicate genes in 1 species (Caenorhabditis briggsae) and their single-copy ortholog in Caenorhabditis elegans. First, using improved genomic sequence and gene model characterization, we confirm that the C. briggsae genome includes 2 complete lin-12 genes, whereas most other genes encoding proteins that participate in the LIN-12 signaling pathway retain a one-to-one orthology with C. elegans. We use CRISPR-mediated genome editing to introduce alleles predicted to cause gain-of-function (gf) or loss-of-function (lf) into each C. briggsae gene and find that the gf mutations uncover functional distinctions not apparent from the lf alleles. Specifically, Cbr-lin-12.1(gf), but not Cbr-lin-12.2(gf), causes developmental defects similar to those observed in Cel-lin-12(gf). In contrast to Cel-lin-12(gf), however, the Cbr-lin-12.1(gf) alleles do not cause dominant phenotypes as compared to the wild type, and the mutant phenotype is observed only when 2 gf alleles are present. Our results demonstrate that gene duplicates can exhibit differential capacities to compensate for each other and to interfere with normal development, and uncover coincident gene duplication and evolution of developmental sensitivity to LIN-12/Notch activity.

"Hurts less, lasts longer"; a qualitative study on experiences of young people receiving high-dose subcutaneous injections of benzathine penicillin G to prevent rheumatic heart disease in New Zealand.

BACKGROUND: Four-weekly intramuscular (IM) benzathine penicillin G (BPG) injections to prevent acute rheumatic fever (ARF) progression have remained unchanged since 1955. A Phase-I trial in healthy volunteers demonstrated the safety and tolerability of high-dose subcutaneous infusions of BPG which resulted in a much longer effective penicillin exposure, and fewer injections. Here we describe the experiences of young people living with ARF participating in a Phase-II trial of SubCutaneous Injections of BPG (SCIP). METHODOLOGY: Participants (n = 20) attended a clinic in Wellington, New Zealand (NZ). After a physical examination, participants received 2% lignocaine followed by 13.8mL to 20.7mL of BPG (Bicillin-LA®; determined by weight), into the abdominal subcutaneous tissue. A Kaupapa Maori consistent methodology was used to explore experiences of SCIP, through semi-structured interviews and observations taken during/after the injection, and on days 28 and 70. All interviews were recorded, transcribed verbatim, and thematically analysed. PRINCIPAL FINDINGS: Low levels of pain were reported on needle insertion, during and following the injection. Some participants experienced discomfort and bruising on days one and two post dose; however, the pain was reported to be less severe than their usual IM BPG. Participants were 'relieved' to only need injections quarterly and the majority (95%) reported a preference for SCIP over IM BPG. CONCLUSIONS: Participants preferred SCIP over their usual regimen, reporting less pain and a preference for the longer time gap between treatments. Recommending SCIP as standard of care for most patients needing long-term prophylaxis has the potential to transform secondary prophylaxis of ARF/RHD in NZ and globally.

Quantifying the fitness effects of resistance alleles with and without anthelmintic selection pressure using Caenorhabditis elegans.

Albendazole (a benzimidazole) and ivermectin (a macrocyclic lactone) are the two most commonly co-administered anthelmintic drugs in mass-drug administration programs worldwide. Despite emerging resistance, we do not fully understand the mechanisms of resistance to these drugs nor the consequences of delivering them in combination. Albendazole resistance has primarily been attributed to variation in the drug target, a beta-tubulin gene. Ivermectin targets glutamate-gated chloride channels (GluCls), but it is unknown whether GluCl genes are involved in ivermectin resistance in nature. Using Caenorhabditis elegans, we defined the fitness costs associated with loss of the drug target genes singly or in combinations of the genes that encode GluCl subunits. We quantified the loss-of-function effects on three traits: (i) multi-generational competitive fitness, (ii) fecundity, and (iii) development. In competitive fitness and development assays, we found that a deletion of the beta-tubulin gene ben-1 conferred albendazole resistance, but ivermectin resistance required the loss of two GluCl genes (avr-14 and avr-15). The fecundity assays revealed that loss of ben-1 did not provide any fitness benefit in albendazole conditions and that no GluCl deletion mutants were resistant to ivermectin. Next, we searched for evidence of multi-drug resistance across the three traits. Loss of ben-1 did not confer resistance to ivermectin, nor did loss of any single GluCl subunit or combination confer resistance to albendazole. Finally, we assessed the development of 124 C. elegans wild strains across six benzimidazoles and seven macrocyclic lactones to identify evidence of multi-drug resistance between the two drug classes and found a strong phenotypic correlation within a drug class but not across drug classes. Because each gene affects various aspects of nematode physiology, these results suggest that it is necessary to assess multiple fitness traits to evaluate how each gene contributes to anthelmintic resistance.

The post-emergence period for denning Polar Bears: phenology and influence on cub survival.

Among polar bears (Ursus maritimus), only parturient females den for extended periods, emerging from maternal dens in spring after having substantially depleted their energy reserves during a fast that can exceed 8 months. Although den emergence coincides with a period of increasing prey availability, polar bears typically do not depart immediately to hunt, but instead remain at the den for up to a month. This delay suggests that there are likely adaptive advantages to remaining at the den between emergence and departure, but the influence of the timing and duration of this post-emergence period on cub survival has not been evaluated previously. We used temperature and location data from 70 denning bears collared within the Southern Beaufort Sea and Chukchi Sea subpopulations to estimate the phenology of the post-emergence period. We evaluated the influence of various spatial and temporal features on duration of the post-emergence period and evaluated the potential influence of post-emergence duration on litter survival early in the spring following denning. For dens that likely contained viable cubs at emergence (n = 56), mean den emergence occurred on 16 March (SE = 1.4 days) and mean departure on 24 March (SE = 1.6 days), with dates typically occurring later in the Chukchi Sea relative to Southern Beaufort Sea and on land relative to sea ice. Mean duration of the post-emergence period was 7.9 days (SE = 1.4) for bears that were observed with cubs later in the spring, which was over 4 times longer than duration of those observed without cubs (1.9 days). Litter survival in the spring following denning (n = 31 dens) increased from 0.5 to 0.9 when duration of the post-emergence period increased by ~4 days and other variables were held at mean values. Our limited sample size and inability to verify cub presence at emergence suggests that future research is merited to improve our understanding of this relationship. Nonetheless, our results highlight the importance of the post-emergence period in contributing to reproductive success and can assist managers in developing conservation and mitigation strategies in denning areas, which will be increasingly important as human activities expand in the Arctic.

Lessons learnt on patient safety in dentistry through a 5-year nationwide database study on iatrogenic harm.

Safe delivery of care is a priority in dentistry, while basic epidemiological knowledge of patient safety incidents is still lacking. The objectives of this study were to (1) classify patient safety incidents related to primary dental care in Denmark in the period 2016-2020 and study the distribution of different types of dental treatment categories where harm occurred, (2) clarify treatment categories leading to "nerve injury" and "tooth loss" and (3) assess the financial cost of patient-harm claims. Data from the Danish Dental Compensation Act (DDCA) database was retrieved from all filed cases from 1st January 2016 until 31st December 2020 pertaining to: (1) The reason why the patient applied for treatment-related harm compensation, (2) the event that led to the alleged harm (treatment category), (3) the type of patient-harm, and (4) the financial cost of all harm compensations. A total of 9069 claims were retrieved, of which 5079 (56%) were found eligible for compensation. The three most frequent categories leading to compensation were "Root canal treatment and post preparation"(n = 2461, 48% of all approved claims), "lack of timely diagnosis and initiation of treatment" (n = 905, 18%) and "surgery" (n = 878, 17%). Damage to the root of the tooth accounted for more than half of all approved claims (54.36%), which was most frequently a result of either parietal perforation during endodontic treatment (18.54%) or instrument fracture (18.89%). Nerve injury accounted for 16.81% of the approved claims. Total cost of all compensation payments was €16,309,310, 41.1% of which was related to surgery (€6,707,430) and 20.4% (€3,322,927) to endodontic treatment. This comprehensive analysis documents that harm permeates all aspects of dentistry, especially in endodontics and surgery. Neglect or diagnostic delays contribute to 18% of claims, indicating that harm does not solely result from direct treatment. Treatment harm inflicts considerable societal costs.

Quantitative tests of albendazole resistance in beta-tubulin mutants.

Benzimidazole (BZ) anthelmintics are among the most important treatments for parasitic nematode infections in the developing world. Widespread BZ resistance in veterinary parasites and emerging resistance in human parasites raise major concerns for the continued use of BZs. Knowledge of the mechanisms of resistance is necessary to make informed treatment decisions and circumvent resistance. Benzimidazole resistance has traditionally been associated with mutations and natural variants in the C. elegans beta-tubulin gene ben-1 and orthologs in parasitic species. However, variants in ben-1 alone do not explain the differences in BZ responses across parasite populations. Here, we examine the roles of five C. elegans beta-tubulin genes (tbb-1, mec-7, tbb-4, ben-1, and tbb-6) to identify the role each gene plays in BZ response. We generated C. elegans strains with a loss of each beta-tubulin gene, as well as strains with a loss of tbb-1, mec-7, tbb-4, or tbb-6 in a genetic background that also lacks ben-1 to test beta-tubulin redundancy in BZ response. We found that only the individual loss of ben-1 conferred a substantial level of BZ resistance, although the loss of tbb-1 was found to confer a small benefit in the presence of albendazole (ABZ). The loss of ben-1 was found to confer an almost complete rescue of animal development in the presence of 30 µM ABZ, likely explaining why no additive effects caused by the loss of a second beta-tubulin were observed. We demonstrate that ben-1 is the only beta-tubulin gene in C. elegans where loss confers substantial BZ resistance.

Getting around the roundworms: Identifying knowledge gaps and research priorities for the ascarids.

The ascarids are a large group of parasitic nematodes that infect a wide range of animal species. In humans, they cause neglected diseases of poverty; many animal parasites also cause zoonotic infections in people. Control measures include hygiene and anthelmintic treatments, but they are not always appropriate or effective and this creates a continuing need to search for better ways to reduce the human, welfare and economic costs of these infections. To this end, Le Studium Institute of Advanced Studies organized a two-day conference to identify major gaps in our understanding of ascarid parasites with a view to setting research priorities that would allow for improved control. The participants identified several key areas for future focus, comprising of advances in genomic analysis and the use of model organisms, especially Caenorhabditis elegans, a more thorough appreciation of the complexity of host-parasite (and parasite-parasite) communications, a search for novel anthelmintic drugs and the development of effective vaccines. The participants agreed to try and maintain informal links in the future that could form the basis for collaborative projects, and to co-operate to organize future meetings and workshops to promote ascarid research.

Quantifying the fitness effects of resistance alleles with and without anthelmintic selection pressure using Caenorhabditis elegans.

Albendazole and ivermectin are the two most commonly co-administered anthelmintic drugs in mass-drug administration programs worldwide. Despite emerging resistance, we do not fully understand the mechanisms of resistance to these drugs nor the consequences of delivering them in combination. Albendazole resistance has primarily been attributed to variation in the drug target, a beta-tubulin gene. Ivermectin targets glutamate-gated chloride channel (GluCl) genes, but it is unknown whether these genes are involved in ivermectin resistance in nature. Using Caenorhabditis elegans, we defined the fitness costs associated with loss of the drug target genes singly or in combinations of the genes that encode GluCl subunits. We quantified the loss-of function effects on three traits: (i) multi-generational competitive fitness, (ii) fecundity, and (iii) development. In competitive fitness and development assays, we found that a deletion of the beta-tubulin gene ben-1 conferred albendazole resistance, but ivermectin resistance required loss of two GluCl genes (avr-14 and avr-15) or loss of three GluCl genes (avr-14, avr-15, and glc-1). The fecundity assays revealed that loss of ben-1 did not provide any fitness benefit in albendazole and that no GluCl deletion mutants were resistant to ivermectin. Next, we searched for evidence of multi-drug resistance across the three traits. Loss of ben-1 did not confer resistance to ivermectin, nor did loss of any single GluCl subunit or combination confer resistance to albendazole. Finally, we assessed the development of 124 C. elegans wild strains across six benzimidazoles and seven macrocyclic lactones to identify evidence of multi-drug resistance between the two drug classes and found a strong phenotypic correlation within a drug class but not across drug classes. Because each gene affects various aspects of nematode physiology, these results suggest that it is necessary to assess multiple fitness traits to evaluate how each gene contributes to anthelmintic resistance.

Adenocarcinoma In Situ of the Uterine Cervix (AIS) Treated by Loop Electrosurgical Excision Procedure Strategy: An Observational Study.

OBJECTIVES: Evaluation of the results of treatment of adenocarcinoma in situ by loop electrosurgical excision procedure and the safety of a conservative strategy. METHODS: Identification of all cases of adenocarcinoma in situ treated by loop electrosurgical excision procedure at our institution and follow-up by a conservative strategy. Completeness of the identification of all cases was secured by data from the National Pathology Registry. The treatment strategy was based on cytologic follow-up performed by a general practitioner and, irrespective of margin status of the cone, only the results of the postoperative surveillance were indicative of further treatment. RESULTS: A total of 224 patients were identified. The overall recurrence rate with a mean follow-up time of 87.8 months was 7.6% (17/224). The recurrence rate in patients with involved margins was significantly higher than in patients with uninvolved margins, 15.7% vs 5.2%, respectively. Six recurrences were diagnosed at first examination 6 months postconization in patients with involved margins. They were treated with hysterectomy in 4 cases and reconization in 1 case. If involvement of margins alone had been an indication of further therapy (hysterectomy or reconization) immediately after conization, the conservative management strategy prevented 46 surgical procedures. Two cases of invasive cancer were diagnosed during follow-up, 150 months and 196 months after primary treatment, and after normal follow-up examinations. These 2 cases must be considered de novo cases and cannot be considered treatment failures. CONCLUSION: The conservative management strategy thus seems safe, and unnecessary surgical procedures were avoided.

CaeNDR, the Caenorhabditis Natural Diversity Resource.

Studies of model organisms have provided important insights into how natural genetic differences shape trait variation. These discoveries are driven by the growing availability of genomes and the expansive experimental toolkits afforded to researchers using these species. For example, Caenorhabditis elegans is increasingly being used to identify and measure the effects of natural genetic variants on traits using quantitative genetics. Since 2016, the C. elegans Natural Diversity Resource (CeNDR) has facilitated many of these studies by providing an archive of wild strains, genome-wide sequence and variant data for each strain, and a genome-wide association (GWA) mapping portal for the C. elegans community. Here, we present an updated platform, the Caenorhabditis Natural Diversity Resource (CaeNDR), that enables quantitative genetics and genomics studies across the three Caenorhabditis species: C. elegans, C. briggsae and C. tropicalis. The CaeNDR platform hosts several databases that are continually updated by the addition of new strains, whole-genome sequence data and annotated variants. Additionally, CaeNDR provides new interactive tools to explore natural variation and enable GWA mappings. All CaeNDR data and tools are accessible through a freely available web portal located at caendr.org.

Genetic Variants That Modify the Neuroendocrine Regulation of Foraging Behavior in C. elegans.

The molecular mechanisms underlying diversity in animal behavior are not well understood. A major experimental challenge is determining the contribution of genetic variants that affect neuronal gene expression to differences in behavioral traits. The neuroendocrine TGF-beta ligand, DAF-7, regulates diverse behavioral responses of Caenorhabditis elegans to bacterial food and pathogens. The dynamic neuron-specific expression of daf-7 is modulated by environmental and endogenous bacteria-derived cues. Here, we investigated natural variation in the expression of daf-7 from the ASJ pair of chemosensory neurons and identified common variants in gap-2, encoding a GTPase-Activating Protein homologous to mammalian SynGAP proteins, which modify daf-7 expression cell-non-autonomously and promote exploratory foraging behavior in a DAF-7-dependent manner. Our data connect natural variation in neuron-specific gene expression to differences in behavior and suggest that genetic variation in neuroendocrine signaling pathways mediating host-microbe interactions may give rise to diversity in animal behavior.

Novel and improved Caenorhabditis briggsae gene models generated by community curation.

BACKGROUND: The nematode Caenorhabditis briggsae has been used as a model in comparative genomics studies with Caenorhabditis elegans because of their striking morphological and behavioral similarities. However, the potential of C. briggsae for comparative studies is limited by the quality of its genome resources. The genome resources for the C. briggsae laboratory strain AF16 have not been developed to the same extent as C. elegans. The recent publication of a new chromosome-level reference genome for QX1410, a C. briggsae wild strain closely related to AF16, has provided the first step to bridge the gap between C. elegans and C. briggsae genome resources. Currently, the QX1410 gene models consist of software-derived gene predictions that contain numerous errors in their structure and coding sequences. In this study, a team of researchers manually inspected over 21,000 gene models and underlying transcriptomic data to repair software-derived errors. RESULTS: We designed a detailed workflow to train a team of nine students to manually curate gene models using RNA read alignments. We manually inspected the gene models, proposed corrections to the coding sequences of over 8,000 genes, and modeled thousands of putative isoforms and untranslated regions. We exploited the conservation of protein sequence length between C. briggsae and C. elegans to quantify the improvement in protein-coding gene model quality and showed that manual curation led to substantial improvements in the protein sequence length accuracy of QX1410 genes. Additionally, collinear alignment analysis between the QX1410 and AF16 genomes revealed over 1,800 genes affected by spurious duplications and inversions in the AF16 genome that are now resolved in the QX1410 genome. CONCLUSIONS: Community-based, manual curation using transcriptome data is an effective approach to improve the quality of software-derived protein-coding genes. The detailed protocols provided in this work can be useful for future large-scale manual curation projects in other species. Our manual curation efforts have brought the QX1410 gene models to a comparable level of quality as the extensively curated AF16 gene models. The improved genome resources for C. briggsae provide reliable tools for the study of Caenorhabditis biology and other related nematodes.

Praziquantel inhibits Caenorhabditis elegans development and species-wide differences might be cct-8-dependent.

Anthelmintic drugs are used to treat parasitic roundworm and flatworm infections in humans and other animals. Caenorhabditis elegans is an established model to investigate anthelmintics used to treat roundworms. In this study, we use C. elegans to examine the mode of action and the mechanisms of resistance against the flatworm anthelmintic drug praziquantel (PZQ), used to treat trematode and cestode infections. We found that PZQ inhibited development and that this developmental delay varies by genetic background. Interestingly, both enantiomers of PZQ are equally effective against C. elegans, but the right-handed PZQ (R-PZQ) is most effective against schistosome infections. We conducted a genome-wide association mapping with 74 wild C. elegans strains to identify a region on chromosome IV that is correlated with differential PZQ susceptibility. Five candidate genes in this region: cct-8, znf-782, Y104H12D.4, Y104H12D.2, and cox-18, might underlie this variation. The gene cct-8, a subunit of the protein folding complex TRiC, has variation that causes a putative protein coding change (G226V), which is correlated with reduced developmental delay. Gene expression analysis suggests that this variant correlates with slightly increased expression of both cct-8 and hsp-70. Acute exposure to PZQ caused increased expression of hsp-70, indicating that altered TRiC function might be involved in PZQ responses. To test if this variant affects development upon exposure to PZQ, we used CRISPR-Cas9 genome editing to introduce the V226 allele into the N2 genetic background (G226) and the G226 allele into the JU775 genetic background (V226). These experiments revealed that this variant was not sufficient to explain the effects of PZQ on development. Nevertheless, this study shows that C. elegans can be used to study PZQ mode of action and resistance mechanisms. Additionally, we show that the TRiC complex requires further evaluation for PZQ responses in C. elegans.

Natural genetic variation in the pheromone production of C. elegans.

From bacterial quorum sensing to human language, communication is essential for social interactions. Nematodes produce and sense pheromones to communicate among individuals and respond to environmental changes. These signals are encoded by different types and mixtures of ascarosides, whose modular structures further enhance the diversity of this nematode pheromone language. Interspecific and intraspecific differences in this ascaroside pheromone language have been described previously, but the genetic basis and molecular mechanisms underlying the variation remain largely unknown. Here, we analyzed natural variation in the production of 44 ascarosides across 95 wild Caenorhabditis elegans strains using high-performance liquid chromatography coupled to high-resolution mass spectrometry. We discovered wild strains defective in the production of specific subsets of ascarosides (e.g., the aggregation pheromone icas#9) or short- and medium-chain ascarosides, as well as inversely correlated patterns between the production of two major classes of ascarosides. We investigated genetic variants that are significantly associated with the natural differences in the composition of the pheromone bouquet, including rare genetic variants in key enzymes participating in ascaroside biosynthesis, such as the peroxisomal 3-ketoacyl-CoA thiolase, daf-22, and the carboxylesterase cest-3. Genome-wide association mappings revealed genomic loci harboring common variants that affect ascaroside profiles. Our study yields a valuable dataset for investigating the genetic mechanisms underlying the evolution of chemical communication.