Egg-laying Defects Research Articles

A small-molecule approach to restore female sterility phenotype targeted by a homing suppression gene drive in the fruit pest Drosophila suzukii.

CRISPR-based gene drives offer promising prospects for controlling disease-transmitting vectors and agricultural pests. A significant challenge for successful suppression-type drive is the rapid evolution of resistance alleles. One approach to mitigate the development of resistance involves targeting functionally constrained regions using multiple gRNAs. In this study, we constructed a 3-gRNA homing gene drive system targeting the recessive female fertility gene Tyrosine decarboxylase 2 (Tdc2) in Drosophila suzukii, a notorious fruit pest. Our investigation revealed only a low level of homing in the germline, but feeding octopamine restored the egg-laying defects in Tdc2 mutant females, allowing easier line maintenance than for other suppression drive targets. We tested the effectiveness of a similar system in Drosophila melanogaster and constructed additional split drive systems by introducing promoter-Cas9 transgenes to improve homing efficiency. Our findings show that genetic polymorphisms in wild populations may limit the spread of gene drive alleles, and the position effect profoundly influences Cas9 activity. Furthermore, this study highlights the potential of conditionally rescuing the female infertility caused by the gene drive, offering a valuable tool for the industrial-scale production of gene drive transgenic insects.

Multiple Subthreshold GPCR Signals Combined by the G-Proteins Gαq and Gαs Activate the Caenorhabditis elegans Egg-Laying Muscles.

Individual neurons or muscle cells express many G-protein-coupled receptors (GPCRs) for neurotransmitters and neuropeptides, yet it remains unclear how cells integrate multiple GPCR signals that all must activate the same few G-proteins. We analyzed this issue in the Caenorhabditis elegans egg-laying system, where multiple GPCRs on muscle cells promote contraction and egg laying. We genetically manipulated individual GPCRs and G-proteins specifically in these muscle cells within intact animals and then measured egg laying and muscle calcium activity. Two serotonin GPCRs on the muscle cells, Gαq-coupled SER-1 and Gαs-coupled SER-7, together promote egg laying in response to serotonin. We found that signals produced by either SER-1/Gαq or SER-7/Gαs alone have little effect, but these two subthreshold signals combine to activate egg laying. We then transgenically expressed natural or designer GPCRs in the muscle cells and found that their subthreshold signals can also combine to induce muscle activity. However, artificially inducing strong signaling through just one of these GPCRs can be sufficient to induce egg laying. Knocking down Gαq and Gαs in the egg-laying muscle cells induced egg-laying defects that were stronger than those of a SER-1/SER-7 double knockout, indicating that additional endogenous GPCRs also activate the muscle cells. These results show that in the egg-laying muscles multiple GPCRs for serotonin and other signals each produce weak effects that individually do not result in strong behavioral outcomes. However, they combine to produce sufficient levels of Gαq and Gαs signaling to promote muscle activity and egg laying.SIGNIFICANCE STATEMENT How can neurons and other cells gather multiple independent pieces of information from the soup of chemical signals in their environment and compute an appropriate response? Most cells express >20 GPCRs that each receive one signal and transmit that information through three main types of G-proteins. We analyzed how this machinery generates responses by studying the egg-laying system of C. elegans, where serotonin and multiple other signals act through GPCRs on the egg-laying muscles to promote muscle activity and egg laying. We found that individual GPCRs within an intact animal each generate effects too weak to activate egg laying. However, combined signaling from multiple GPCR types reaches a threshold capable of activating the muscle cells.

Serotonin signals through postsynaptic Gαq, Trio RhoGEF, and diacylglycerol to promote Caenorhabditis elegans egg-laying circuit activity and behavior.

Activated Gαq signals through phospholipase-Cβ and Trio, a Rho GTPase exchange factor (RhoGEF), but how these distinct effector pathways promote cellular responses to neurotransmitters like serotonin remains poorly understood. We used the egg-laying behavior circuit of Caenorhabditis elegans to determine whether phospholipase-Cβ and Trio mediate serotonin and Gαq signaling through independent or related biochemical pathways. Our genetic rescue experiments suggest that phospholipase-Cβ functions in neurons while Trio Rho GTPase exchange factor functions in both neurons and the postsynaptic vulval muscles. While Gαq, phospholipase-Cβ, and Trio Rho GTPase exchange factor mutants fail to lay eggs in response to serotonin, optogenetic stimulation of the serotonin-releasing HSN neurons restores egg laying only in phospholipase-Cβ mutants. Phospholipase-Cβ mutants showed vulval muscle Ca2+ transients while strong Gαq and Trio Rho GTPase exchange factor mutants had little or no vulval muscle Ca2+ activity. Treatment with phorbol 12-myristate 13-acetate that mimics 1,2-diacylglycerol, a product of PIP2 hydrolysis, rescued egg-laying circuit activity and behavior defects of Gαq signaling mutants, suggesting both phospholipase-C and Rho signaling promote synaptic transmission and egg laying via modulation of 1,2-diacylglycerol levels. 1,2-Diacylglycerol activates effectors including UNC-13; however, we find that phorbol esters, but not serotonin, stimulate egg laying in unc-13 and phospholipase-Cβ mutants. These results support a model where serotonin signaling through Gαq, phospholipase-Cβ, and UNC-13 promotes neurotransmitter release, and that serotonin also signals through Gαq, Trio Rho GTPase exchange factor, and an unidentified, phorbol 12-myristate 13-acetate-responsive effector to promote postsynaptic muscle excitability. Thus, the same neuromodulator serotonin can signal in distinct cells and effector pathways to coordinate activation of a motor behavior circuit.

Cellular Expression and Functional Roles of All 26 Neurotransmitter GPCRs in the C. elegans Egg-Laying Circuit.

Maps of the synapses made and neurotransmitters released by all neurons in model systems, such as Caenorhabditis elegans have left still unresolved how neural circuits integrate and respond to neurotransmitter signals. Using the egg-laying circuit of C. elegans as a model, we mapped which cells express each of the 26 neurotransmitter GPCRs of this organism and also genetically analyzed the functions of all 26 GPCRs. We found that individual neurons express many distinct receptors, epithelial cells often express neurotransmitter receptors, and receptors are often positioned to receive extrasynaptic signals. Receptor knockouts reveal few egg-laying defects under standard laboratory conditions, suggesting that the receptors function redundantly or regulate egg-laying only in specific conditions; however, increasing receptor signaling through overexpression more efficiently reveals receptor functions. This map of neurotransmitter GPCR expression and function in the egg-laying circuit provides a model for understanding GPCR signaling in other neural circuits.SIGNIFICANCE STATEMENT Neurotransmitters signal through GPCRs to modulate activity of neurons, and changes in such signaling can underlie conditions such as depression and Parkinson's disease. To determine how neurotransmitter GPCRs together help regulate function of a neural circuit, we analyzed the simple egg-laying circuit in the model organism C. elegans We identified all the cells that express every neurotransmitter GPCR and genetically analyzed how each GPCR affects the behavior the circuit produces. We found that many neurotransmitter GPCRs are expressed in each neuron, that neurons also appear to use these receptors to communicate with other cell types, and that GPCRs appear to often act redundantly or only under specific conditions to regulate circuit function.

Modulation of serotonin signaling by the putative oxaloacetate decarboxylase FAHD-1 in Caenorhabditis elegans.

Human fumarylacetoacetate hydrolase (FAH) domain containing protein 1 (FAHD1) is a mitochondrial oxalocatate decarboxylase, the first of its kind identified in eukaryotes. The physiological role of FAHD1 in other eukaryotes is still poorly understood. In C. elegans loss of the FAHD1 ortholog FAHD-1 was reported to impair mitochondrial function, locomotion and egg-laying behavior, yet the underlying mechanisms remained unclear. Using tissue-specific rescue of fahd-1(-) worms, we find that these phenotypic abnormalities are at least in part due to fahd-1’s function in neurons. Moreover, we show that egg-laying defects in fahd-1(-) worms can be fully rescued by external dopamine administration and that depletion of fahd-1 expression induces expression of several enzymes involved in serotonin biosynthesis. Together, our results support a role for fahd-1 in modulating serotonin levels and suggest this protein as a novel link between metabolism and neurotransmitter signaling in the nervous system. Finally, we propose a model to explain how a metabolic defect could ultimately lead to marked changes in neuronal signaling.

Serotonin and neuropeptides are both released by the HSN command neuron to initiate Caenorhabditis elegans egg laying.

Neurons typically release both a small-molecule neurotransmitter and one or more neuropeptides, but how these two types of signal from the same neuron might act together remains largely obscure. For example, serotonergic neurons in mammalian brain express the neuropeptide Substance P, but it is unclear how this co-released neuropeptide might modulate serotonin signaling. We studied this issue in C. elegans, in which all serotonergic neurons express the neuropeptide NLP-3. The serotonergic Hermaphrodite Specific Neurons (HSNs) are command motor neurons within the egg-laying circuit which have been shown to release serotonin to initiate egg-laying behavior. We found that egg-laying defects in animals lacking serotonin were far milder than in animals lacking HSNs, suggesting that HSNs must release other signal(s) in addition to serotonin to stimulate egg laying. While null mutants for nlp-3 had only mild egg-laying defects, animals lacking both serotonin and NLP-3 had severe defects, similar to those of animals lacking HSNs. Optogenetic activation of HSNs induced egg laying in wild-type animals, and in mutant animals lacking either serotonin or NLP-3, but failed to induce egg laying in animals lacking both. We recorded calcium activity in the egg-laying muscles of animals lacking either serotonin, NLP-3, or both. The single mutants, and to a greater extent the double mutant, showed muscle activity that was uncoordinated and unable to expel eggs. Specifically, the vm2 muscles cells, which are direct postsynaptic targets of the HSN, failed to contract simultaneously with other egg-laying muscle cells. Our results show that the HSN neurons use serotonin and the neuropeptide NLP-3 as partially redundant co-transmitters that together stimulate and coordinate activity of the target cells onto which they are released.

Pleiotropic Effects of Taurine on Nematode Model for Down Syndrome.

Taurine is traditionally used to treat Down Syndrome (DS); however, the actual foundation for this treatment is not well understood. DS patients suffer from disturbance of the proteostasis network (PN) due to aberrant calcium signaling, which eventually causes endoplasmic reticulum stress (ERS). Taurine has been suggested to play a role in modulating calcium homeostasis and ERS. This study examined whether taurine affects DS symptoms using C. elegans- a DS model in which calcineurins, Ca2+/calmodulin-dependent protein phosphatase is mutated to null. The DS nematode model has short body length, slow growth, fertility defects, serotonin-resistant egg-laying defects, and faulty thermal sensing. This study focused on whether taurine may ameliorate the severity of DS at the whole-body level, including reduction in ERS. When treated with taurine, DS nematodes appeared to have lower levels of ERS and phenotypes closer to the wild type. DS nematodes also showed improved egg laying efficiency and thermal sensing index comparable to the wild type. Our findings offer a new perspective on the effectiveness of taurine in treating DS and designing therapeutic strategies to lower ERS and restore disrupted PN.

Combining bioinformatics, cheminformatics, functional genomics and whole organism approaches for identifying epigenetic drug targets in Schistosoma mansoni

Schistosomiasis endangers the lives of greater than 200 million people every year and is predominantly controlled by a single class chemotherapy, praziquantel (PZQ). Development of PZQ replacement (to combat the threat of PZQ insensitivity/resistance arising) or combinatorial (to facilitate the killing of PZQ-insensitive juvenile schistosomes) chemotherapies would help sustain this control strategy into the future. Here, we re-categorise two families of druggable epigenetic targets in Schistosoma mansoni, the histone methyltransferases (HMTs) and the histone demethylases (HDMs). Amongst these, a S. mansoni Lysine Specific Demethylase 1 (SmLSD1, Smp_150560) homolog was selected for further analyses. Homology modelling of SmLSD1 and in silico docking of greater than four thousand putative inhibitors identified seven (L1 – L7) showing more favourable binding to the target pocket of SmLSD1 vs Homo sapiens HsLSD1; six of these seven (L1 – L6) plus three structural analogues of L7 (L8 – L10) were subsequently screened against schistosomula using the Roboworm anthelmintic discovery platform. The most active compounds (L10 - pirarubicin > L8 – danunorubicin hydrochloride) were subsequently tested against juvenile (3 wk old) and mature (7 wk old) schistosome stages and found to impede motility, suppress egg production and affect tegumental surfaces. When compared to a surrogate human cell line (HepG2), a moderate window of selectivity was observed for the most active compound L10 (selectivity indices - 11 for schistosomula, 9 for juveniles, 1.5 for adults). Finally, RNA interference of SmLSD1 recapitulated the egg-laying defect of schistosomes co-cultivated in the presence of L10 and L8. These preliminary results suggest that SmLSD1 represents an attractive new target for schistosomiasis; identification of more potent and selective SmLSD1 compounds, however, is essential. Nevertheless, the approaches described herein highlight an interdisciplinary strategy for selecting and screening novel/repositioned anti-schistosomals, which can be applied to any druggable (epigenetic) target encoded by the parasite's genome.

Identification of small-molecule ion channel modulators in C. elegans channelopathy models

Ion channels are important therapeutic targets, but the discovery of ion channel drugs remains challenging due to a lack of assays that allow high-throughput screening in the physiological context. Here we report C. elegans phenotype-based methods for screening ion channel drugs. Expression of modified human ether-a-go-go-related gene (hERG) potassium channels in C. elegans results in egg-laying and locomotive defects, which offer indicators for screening small-molecule channel modulators. Screening in worms expressing hERGA561V, which carries a trafficking-defective mutation A561V known to associate with long-QT syndrome, identifies two functional correctors Prostratin and ingenol-3,20-dibenzoate. These compounds activate PKCε signaling and consequently phosphorylate S606 at the pore region of the channel to promote hERGA561V trafficking to the plasma membrane. Importantly, the compounds correct electrophysiological abnormalities in hiPSC-derived cardiomyocytes bearing a heterozygous CRISPR/Cas9-edited hERGA561V. Thus, we have developed an in vivo high-throughput method for screening compounds that have therapeutic potential in treating channelopathies.

Caenorhabditis elegans respond to high-glucose diets through a network of stress-responsive transcription factors.

High-glycemic-index diets, as well as a sedentary lifestyle are considered as determinant factors for the development of obesity, type 2 diabetes, and cardiovascular diseases in humans. These diets have been shown to shorten the life span of C. elegans in a manner that is dependent on insulin signaling, but the participation of other signaling pathways have not been addressed. In this study, we have determined that worms fed with high-glucose diets show alterations in glucose content and uptake, triglyceride content, body size, number of eggs laid, egg-laying defects, and signs of oxidative stress and accelerated aging. Additionally, we analyzed the participation of different key regulators of carbohydrate and lipid metabolism, oxidative stress and longevity such as SKN-1/NRF2, HIF-1/HIF1α, SBP-1/SREBP, CRH-1/CREB, CEP-1/p53, and DAF-16/FOXO, in the reduction of lifespan in glucose-fed worms.

The Good, the Bad, and the Lethal: Gene Expression and Metabolomics Reveal Physiological Mechanisms Underlying Chronic Thermal Effects in Mayfly Larvae (Neocloeon triangulifer)

Temperature dictates the performance of aquatic ectotherms. However, the physiological and biochemical processes that drive thermally-mediated life history patterns (and limits) remain poorly understood because they are rarely studied simultaneously. In our previous work, we have established life history outcomes (e.g. survivorship, development time, growth rates and fitness) in mayflies (Neocloeon triangulifer) reared at static temperatures ranging from 14°C - 30°C at 2°C intervals. In this study, we conducted biochemical measurements (RT-qPCR of select genes and targeted, quantitative metabolomic profiling) on N. triangulifer mature larvae reared at temperatures associated with excellent survival and fitness (22-24°C), compromised survival and fitness (28°C), and chronic lethality (30°C -larvae survived for a few weeks but failed to emerge to adulthood). Patterns of gene expression were similar to those observed in acute ramping experiments reported previously: larvae reared at 30°C resulted in significant upregulation in the thermally responsive gene HEAT SHOCK PROTEIN 90 (HSP90) but no significant changes in hypoxia responsive genes (EGG LAYING DEFECTIVE 9 (EGL-9) and LACTATE DEHYDROGENASE (LDH)). Additionally, primers for genes associated with energy: INSULIN RECEPTOR (IR), mechanistic TARGET OF RAPAMYCIN (mTOR) and TREHALOSE 6 PHOSPHATE SYNTHASE (T6PS) were developed for this study. IR and mTOR were significantly upregulated while T6PS showed trend of downregulation in larvae reared at 30°C. Metabolomic profiles revealed general depletion of lipids and acylcarnitines in larvae exposed to chronic thermal stress, suggesting that larvae were energetically challenged despite continuous access to food. For example, concentrations of lysoPhosphatidylcholine (lysoPC) a C20:3 decreased as fitness decreased with increasing temperature (2.3 fold and 2.4 fold at 28 and 30°C relative to controls). Tissue concentrations of the biogenic amine histamine increased 2.1 and 3.1 fold with increasing temperature, and were strongly and negatively correlated with performance. Thus, both histamine and lysoPC a C20:3 are potential biomarkers of thermal stress. Taken together, our results primarily associate energetic challenge with thermally mediated fitness reduction in N. triangulifer.

FLP-1 neuropeptides modulate sensory and motor circuits in the nematode Caenorhabditis elegans.

Parasitic nematodes infect over one quarter of the population worldwide, causing morbidity in over one billion people. Current anthelmintic drugs are beginning to lose effectiveness due to the presence of resistant strains. We are interested in the role of neuropeptides, which regulate behaviors in all organisms, as another possible target for anthelmintic drugs. FMRFamide-related peptides (FaRPs) are a family of neuropeptides that are conserved throughout the animal kingdom. In particular, nematodes contain the largest family of FaRPs identified thus far and many of these FaRPs are identical among different nematode species; FaRPs in nematodes are collectively referred to as FLPs (FMRFamide-like peptides). However, little is known about the function of these FLPs. We are using the non-parasitic nematode Caenorhabditis elegans as a model for examining FLPs in nematodes. C. elegans contains at least 31 flp genes that encode 72 potential FLPs. Among the flp genes, flp-1 is one of the few that is universally found in nematodes. FLP-1 neuropeptides were previously reported to be involved in sensory and motor functions. However, previous alleles of flp-1 also disrupted a neighboring gene, daf-10. To understand the phenotypes of flp-1, new alleles that specifically disrupt flp-1 were characterized. The previously reported locomotory and egg-laying defects were found to be due to loss of flp-1, while the osmolarity defect is due to loss of daf-10. In addition, loss of flp-1 and daf-10 both cause several phenotypes that increase in severity in the double mutants by disrupting different neurons in the neural circuits.

The octopamine receptor octß2R is essential for ovulation and fertilization in the fruit fly Drosophila melanogaster.

The biogenic monoamine octopamine is essential for ovulation and fertilization in insects. Release of this hormone from neurons in the thoracoabdominal ganglion triggers ovulation and sperm release from the spermathecae. Here we show that the effects of octopamine on ovulation are mediated by at least two different octopamine receptors. In addition to the Oamb receptor that is present in the epithelium of the oviduct, the octß2R receptor is essential for ovulation and fertilization. Octß2R is widely expressed in the female reproductive tract. Most prominent is expression in the oviduct muscle and the spermathecae. Animals deficient in expression of the receptor show a severe egg-laying defect. The corresponding females have a much larger ovary that is caused by egg retention in the ovary. Moreover, the very few laid eggs are not fertilized, indicating problems in the process of sperm delivery. We assume that octß2R acts in a similar way as ß2-adrenoreceptors in smooth muscles, were activation of this receptor induces an increase in cAMP levels that lead to relaxation of the muscle. Taken together, our findings show that octopaminergic control of ovulation and fertilization is more complex than anticipated and that various receptors located in different cells act together to enable a well-orchestrated activity of the female reproductive system in response to copulation.

Identification of a cell adhesion molecule involved in negative regulation of presynaptic assembly in C.elegans

Synapses are asymmetric structures that are specialized for neuronal signal transduction. The molecular mechanisms of how the assembly of presynaptic components is initiated and regulated remain unknown. In C. elegans hermaphrodite‐specific motor neurons HSN, SYD‐1 (a conserved protein with PDZ, C2 and RhoGAP domains) and SYD‐2 (a Liprin‐α adapter protein) are essential for the assembly of presynaptic components at synaptic region. Loss‐of‐function of syd‐1 or syd‐2 causes failure in the formation of presynaptic structure at normal synaptic location, which results in abnormal egg‐laying behavior. To further understand the molecular mechanisms, we carried out genetic suppressor screen of syd‐1. We identified a mutant that restored egg‐laying defects and partially improved the localization of synaptic vesicle markers at synaptic sites in HSN in syd‐1 loss‐of‐function animals. The mutant weakly suppressed egg‐laying defects and synaptic defects in HSN in syd‐2 loss‐of‐function. The responsible gene encodes a non‐classical cadherin protein. Future characterization of the mutant may contribute to our understanding of molecular mechanisms of presynaptic assembly.

Functional analysis of the ATP-binding cassette (ABC) transporter gene family of Tribolium castaneum

BackgroundThe ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. Most are integral membrane proteins that transport a broad spectrum of substrates across lipid membranes. In insects, ABC transporters are of special interest because of their role in insecticide resistance.ResultsWe have identified 73 ABC transporter genes in the genome of T. castaneum, which group into eight subfamilies (ABCA-H). This coleopteran ABC family is significantly larger than those reported for insects in other taxonomic groups. Phylogenetic analysis revealed that this increase is due to gene expansion within a single clade of subfamily ABCC. We performed an RNA interference (RNAi) screen to study the function of ABC transporters during development. In ten cases, injection of double-stranded RNA (dsRNA) into larvae caused developmental phenotypes, which included growth arrest and localized melanization, eye pigmentation defects, abnormal cuticle formation, egg-laying and egg-hatching defects, and mortality due to abortive molting and desiccation. Some of the ABC transporters we studied in closer detail to examine their role in lipid, ecdysteroid and eye pigment transport.ConclusionsThe results from our study provide new insights into the physiological function of ABC transporters in T. castaneum, and may help to establish new target sites for insect control.

Quantum dot nanoparticles affect the reproductive system of Caenorhabditis elegans

Quantum dots (QDs) are an increasingly important class of nanoparticle, but little ecotoxicological data for QDs has been published to date. The effects of mercaptosuccinic acid (MSA)-capped QDs (QDs-MSA) and equivalent concentrations of cadmium (Cd) from cadmium chloride on growth and reproduction of the nematode Caenorhabditis elegans (Rhabditidae) were assessed in laboratory experiments. Growth from larvae to adults of C. elegans was unaffected by exposure to 1 µM fluorescent QDs-MSA, but adults produced more embryos and laid them prematurely. Furthermore, C. elegans exposed to QDs-MSA (1 µM) showed a high percentage of embryo mortality (19.2 ± 0.5, p < 0.001, percentage ± standard deviation) compared with unexposed nematodes (11.6 ± 0.4). An egg-laying defect phenotype was also observed at high frequency in response to 1 µM QDs-MSA exposure (38.3 ± 3.6%, p < 0.01; control 10.0 ± 2.2%). This resulted in a reduced mean life span (20.5 ± 1.1 d, p < 0.05) compared with the control (24.6 ± 1.0 d). Cadmium also caused reduced life span in C. elegans, but a low incidence of egg-laying defects was observed, suggesting that Cd and QDs-MSA affected C. elegans by different mechanisms. Furthermore, egg-laying defects caused by QDs-MSA responded to the addition of the anticonvulsant ethosuximide and to a lesser extent to the neurotransmitter serotonin, suggesting that QDs-MSA might have disrupted motor neurons during the reproduction process.

Characterization of the Nematicidal Activity of Natural Honey

Antimicrobial activities of honey against bacteria and fungi are extensively reported in the scientific literature. However, its nematicidal potential has not been characterized so far. This study examined the effect of natural honey on model nematode Caenorhabditis elegans and analyzed the honey component(s) responsible for nematicidal activity. Characterization of honey-treated C. elegans was done using fluorescence and phase contrast microscopy. Egg-laying and egg-hatching defects of honey-treated C. elegans were studied. For identification of nematicidal component(s), bioactivity-directed fractionation of honey samples was carried out using dialysis, ultrafiltration, chromatographic, and spectroscopic techniques. Natural honeys of different floral sources showed nematicidal activity against different developmental stages of C. elegans. The nematicidal components of honey induced cell death in intestinal lumen and gonads of C. elegans as revealed by microscopy. The nematicidal action of honey was found to be due to reproductive anomaly as manifested by defects in egg-laying and -hatching by C. elegans. Honey with concentration as low as 0.03% exerted profound egg-laying defects, whereas 6% honey showed defects in egg hatching. The major sugar components of honey were not involved in observed nematicidal activity. The bioactive components responsible for anti-C. elegans activity were found in the 2-10 kDa fraction of honey, which was resolved into ∼25 peaks by reverse phase HPLC. LC-MS followed by further spectroscopic characterization revealed a glycoconjugate with the molecular mass of 5511 as the major nematicidal component of honey.

A mutation in a CLC anion channel alters serotonergic neuronal activity in C. elegans

Egg‐laying in C. elegans is controlled by the HSN neurons, which release serotonin to activate egg‐laying muscles. The dominant egl‐42(n995) mutation prevents serotonin release and inhibits egg‐laying. Intracellular Ca2+ imaging demonstrated that HSN activity is reduced in egl‐42 mutants. Whole genome sequencing revealed that egl‐42(n995) is a point mutation in the clh‐3 gene, which encodes the CLC anion channel splice variants CLH‐3a and CLH‐3b. Overexpression of both wild‐type and mutant clh‐3 phenocopies the egl‐42(n995) egg‐laying defect, while clh‐3 deletion leads to hyperactive egg‐laying. The n995 allele is a valine to alanine mutation that is located in membrane helix P, which contributes to the monomer‐monomer interface in CLC homodimers. This residue is highly conserved in all human CLC proteins except CLC‐Ka and CLC‐Kb. CLH‐3a and CLH‐3b mutant channels exhibit increased current amplitude, a strongly depolarized activation voltage, and more rapid voltage dependent activation kinetics. CLH‐3a and CLH‐3b mutants also exhibit loss of pre‐potentiation behavior and greatly reduced sensitivity to inhibition by GCK‐3 kinase, respectively. Our results demonstrate 1) that CLC helix P plays an important role in controlling channel voltage sensitivity and 2) that clh‐3 encoded channels likely regulate HSN membrane potential and hence Ca2+ entry and neuronal excitability.

STR-33, a Novel G Protein-coupled Receptor That Regulates Locomotion and Egg Laying in Caenorhabditis elegans

Despite their predicted functional importance, most G protein-coupled receptors (GPCRs) in Caenorhabditis elegans have remained largely uncharacterized. Here, we focused on one GPCR, STR-33, encoded by the str-33 gene, which was discovered through a ligand-based screening procedure. To characterize STR-33 function, we performed UV-trimethylpsolaren mutagenesis and isolated an str-33-null mutant. The resulting mutant showed hypersinusoidal movement and a hyperactive egg-laying phenotype. Two types of egg laying-related mutations have been characterized: egg laying-deficient (Egl-d) and hyperactive egg laying (Egl-c). The defect responsible for the egg laying-deficient Egl-d phenotype is related to Gα(q) signaling, whereas that responsible for the opposite, hyperactive egg-laying Egl-c phenotype is related to Gα(o) signaling. We found that the hyperactive egg-laying defect of the str-33(ykp001) mutant is dependent on the G protein GOA-1/Gα(o). Endogenous acetylcholine suppressed egg laying in C. elegans via a Gα(o)-signaling pathway by inhibiting serotonin biosynthesis or release from the hermaphrodite-specific neuron. Consistent with this, in vivo expression of the serotonin biosynthetic enzyme, TPH-1, was up-regulated in the str-33(ykp001) mutant. Taken together, these results suggest that the GPCR, STR-33, may be one of the neurotransmitter receptors that regulates locomotion and egg laying in C. elegans.

A Novel Auxiliary Subunit Critical to BK Channel Function inCaenorhabditis elegans

The BK channel is a Ca²+- and voltage-gated potassium channel with many important physiological functions. To identify proteins important to its function in vivo, we screened for Caenorhabditis elegans mutants that suppressed a lethargic phenotype caused by expressing a gain-of-function (gf) isoform of the BK channel α-subunit SLO-1. BKIP-1 (for BK channel interacting protein), a small peptide with no significant homology to any previously characterized molecules, was thus identified. BKIP-1 and SLO-1 showed similar expression and subcellular localization patterns and appeared to interact physically through discrete domains. bkip-1 loss-of-function (lf) mutants phenocopied slo-1(lf) mutants in behavior and synaptic transmission and suppressed the lethargy, egg-laying defect, and deficient neurotransmitter release caused by SLO-1(gf). In heterologous expression systems, BKIP-1 decreased the activation rate and shifted the conductance-voltage relationship of SLO-1 in a Ca²+-dependent manner and increased SLO-1 surface expression. Thus, BKIP-1 is a novel auxiliary subunit critical to SLO-1 function in vivo.