Ghosh, Rajarshi et al. (2013) International Worm Meeting "Evolution of avermectin resistance in C. briggsae."

Kruglyak, Leonid, Thomas, Cristel, Wang, Wei, Jovelin, Richard, Cutter, Asher, Ghosh, Rajarshi

[International Worm Meeting, 2013]

Several nematode species have evolved resistance to the widely used anthelmintic avermectins (AVM). AVM is produced naturally by S.avermitilis, a ubiquitous soil bacterium. As many nematodes spend part of their life cycle in contact with soil, they are likely to encounter S.avermitilis. Widespread AVM resistance may be a result of different nematode species' ability to counter a common selective pressure, namely the toxins produced by S. avermitilis. To test this hypothesis we surveyed AVM resistance in diverse nematode species. We found that resistance to AVM and to S. avermitilis was prevalent in this phylum. To identify the genetic basis of natural AVM resistance we focused on C. briggsae. We found that two divergent isolates of C. briggsae differed significantly in their responses to AVM. Using QTL mapping approach with these two strains , we identified a significant locus on Chromosome II underlying responses to AVM. We also surveyed 50 isolates of C. briggsae for responses to AVM and found that they exhibit wide variation. The pattern of variation in responses to AVM correlated significantly with the observed phylogeographic pattern in C. briggsae, with temperate isolates being more likely to be resistant than tropical ones. To gain insights into the evolution AVM resistance in C. briggsae, we obtained whole genome sequences of these isolates. Using this data we confirmed that glc-1, the causative gene for natural AVM resistance in C. elegans, is the result of a duplication of another GluCl subunit in the elegans lineage. Thus glc-1 is absent in C. briggsae suggesting that the genetic mechanisms of natural resistance to AVM in C. briggsae are likely different from C. elegans. The sequence data will help us determine if variation in candidate targets for AVM correlate with the pattern of resistance in C. briggsae and map the genetic basis of differences in responses to AVM in C. briggsae .

Ghosh, Rajarshi et al. (2013) International Worm Meeting "Genetics of praziquantel resistance in C. elegans."

Ghosh, Rajarshi, Levinson, Anya, Tenenbaum, Conrad, Kruglyak, Leonid

[International Worm Meeting, 2013]

The anthelmintic praziquantel (PRZ) is widely used for treating parasitic flatworm infections in human. It is the most-used drug for treating schistosomasis, a debilitating fluke-borne disease. Despite this prevalence, the target of PRZ is still unknown. Dependence on a single drug with unknown target, for a disease affecting 240 million people worldwide, makes the problem of emergence of drug resistance a special concern. The target and mechanism of action of PRZ has been elusive perhaps because it is thought that nematodes are unaffected by this drug. Indeed we found that the laboratory strain N2 is resistant to PRZ relative to other wild isolates of C. elegans. However, we observed that, as in flatworms, PRZ induces spastic paralysis in several nematode species and wild isolates of C. elegans. To identify the genetic targets of PRZ, we mutagenized N2 and Hawaiian (CB4856) strains and selected mutants resistant to high dose of PRZ. We isolated nine and two mutants in the N2 and CB4856 background respectively. Through whole genome sequencing, complementation tests, RNAi and transgenic rescue we identified the first genes contributing to PRZ resistance in C.elegans. Interestingly different genes in the N2 and CB4856 background conferred PRZ resistance. Tissue specific knockdown experiments and epistatsis analysis suggested that PRZ disrupts osmoregulation in C. elegans. We also found that CB4856 animals were significantly more sensitive to PRZ than N2 animals. To identify genetic basis of individual differences in responses to PRZ, we took a quantitative trait loci mapping approach and identified a significant locus on chromosome IV. Currently we are using transgenic rescue to fine map the QTL and testing the interactions between the QTL and the genes identified through mutagenesis. Using classical and quantitative genetic approaches and by studying wild isolates beyond the laboratory strain, we uncovered novel insights into mechanism of action of PRZ .

Ghosh, D. D. et al. (2015) International Worm Meeting "Neuroendocrine reinforcement of a dynamic multisensory decision."

Nitabach, M.N., Sanders, T., Cohen, N., Hong, S., Koelle, M.R., Chase, D.L., Ghosh, D. D.

[International Worm Meeting, 2015]

To navigate complex natural environments containing both dangerous and valuable items, animals must make economic decisions on the basis of information transduced by multiple senses. However, detailed underlying neural mechanisms of multisensory decision making remain poorly understood. Here we confronted worms with a multisensory decision in which the reward of food must be balanced with the threat of desiccation imposed by a hyperosmotic barrier intervening between the worm and a source of food odor. We find that this decision is modulated by food deprivation. To identify neural substrates underlying this decision, we focused on the RIM interneuron, which is advantageously positioned to transduce integrated multisensory information into locomotor outputs. Consistent with this hypothesis, we find that the activation of a neuropeptide receptor in RIM sets the balance of threat and reward in this decision, with greater receptor activation biasing the worm against crossing the dangerous barrier. Unexpectedly, however, RIM controls the decision not by synaptic signaling to the downstream premotor command circuit, but rather by extrasynaptic aminergic signaling directly onto the primary osmosensory neuron to tune its sensitivity. Additionally, our results suggest that this neuromodulator relay is suppressed in food deprived states, thereby providing a link between internal state, neural network activity, and decision making. Finally, to characterize the complex and dynamic interplay between neuromodulator activity, neuron state, and behavior in the decision making arena, we reproduced the paradigm in silico [1]. Computational modeling revealed how non-linear sensory integration in RIM modulates neuromodulator circuit activity to implement the decision. Taken together, these studies reveal a cellular and molecular mechanism for a dynamic multisensory decision. Intriguingly, our results identified organizational circuit principles conserved between mammalian and C. elegans multisensory decision making. Therefore our studies have broad implications for understanding principles underlying multisensory decision making in Metazoans.1Sanders, T., Ghosh, D.D., Nitabach, M.N., and Cohen, N. "Nonlinear sensory integration in C. elegans: a computational model." International C. elegans meeting.

Ghosh, Rajarshi et al. (2011) International Worm Meeting "Quantitative genetic dissection of a behavioral sequence in C. elegans."

Kruglyak, Leonid, Ryu, William S., Ghosh, Rajarshi

[International Worm Meeting, 2011]

Individuals within species exhibit heritable variation in sub-steps of a behavioral sequence. How are different parts of a behavioral sequence parsed at the genetic level? What genetic changes and architecture allow for natural variation in substeps of a behavioral sequence? To address these questions we transiently raised the local temperature around a worm and recorded several aspects of the resulting escape response. Upon sensation of a thermal impulse worms exhibit a behavioral sequence in which they enter a pause state followed by reversals, omega turn and resumption of forward movement. We characterized several aspects of the escape response for the laboratory strain (N2) and a wild isolate (CB4856) of C. elegans for stimuli resulting in 0.3, 0.8, 3 or 7.5degC rise in temperature. We found that various aspects of the response to 0.3degC increase in temperature exhibited the greatest difference between these strains. We employed a quantitative trait loci (QTL) mapping approach and measured several traits including but not limited to speeds at every 0.18 seconds during the assay, probability of transition between various states, duration and number of reversals in recombinant inbred lines (RIAIL) made from N2 and CB4856. We identified several QTL suggesting different aspects of the escape response are under distinct genetic control e.g. different phases of speed-profile during the assay were genetically separable. npr-1, shown to be responsible for variation in speed between N2 and CB4856, contributes to variation in pre-stimulus speed and deceleration. QTL on chromosome(chr) IV contributes to variation in acceleration to maximum speed after the thermal impulse. Probability of responding by reversal is regulated by additive QTL on chr V and X. For this trait, F1 worms resulting from reciprocal crosses between N2 and CB4856, responded like CB4856 suggesting molecular loss of function allele(s) in N2. Using introgression lines we narrowed down the QTL interval on chr X to ~90 genes. To determine if the pattern of genetic correlation between different aspects of escape behavior in the RIAILs reflect the broader population of C.elegans we quantified responses of 70 wild isolates of distinct haplotypes. Preliminary analysis suggests abundant phenotypic variation and a pattern of genetic correlation among the different sub-behaviors similar to RIAILs.

Ghosh Roy, A. et al. (2009) International Worm Meeting "Dissecting Calcium/cAMP mediated axon regeneration pathways."

Wu, Z., Chisholm, A.D., Ghosh Roy, A., Goncharov, A., Jin, Y.

[International Worm Meeting, 2009]

We are interested in the molecular pathways that promote the regenerative response of axons after injury. Injury of axons of cultured neurons causes a transient elevation of intracellular calcium. Pharmacological elevation of calcium and cAMP are long known to enhance the ability of axons to regenerate in vitro and in vivo. However the molecular mechanisms that govern the Ca/cAMP mediated axonal regeneration are not clearly understood. We previously reported that the lateral touch neurons ALM and PLM show robust regenerative responses after injury (Wu et al., PNAS, 2007). We report here that calcium and cAMP play critical roles in the regenerative responses of these neurons. Using genetically encoded Ca sensors we have found that laser axotomy triggers a rapid local elevation of intracellular calcium that propagates bidirectionally away from the axotomy site. The amplitude of this calcium transient correlates with the extent of total axonal regrowth. Further, gain of function in the voltage-gated calcium channel, egl-19 increases the amplitude of the calcium transient and accelerates regenerative growth, suggesting calcium levels are a critical determinant of regrowth kinetics. We find that genetic elevation of calcium enhances regeneration in several ways. The injured axon enters the regeneration phase earlier compared to controls, extends faster, and more often fuses with the severed distal fragment. Our ultrastructural analysis shows that in these cases the regrowing axon has physically fused membranes with the distal fragment. This fusion process appears not to require known fusogens such as EFF-1 or AFF-1. Mutations that chronically elevate neuronal cAMP promote regeneration in a similar manner with the exception that the regenerating process also has an increased tendency to extend ventral synaptic branches. Reduction of neuronal cAMP by overexpression of PDE-4 blocked this ventral branch regrowth, indicating that cAMP functions cell autonomously to promote growth. Activation of protein kinase A has similar effects on regeneration, suggesting PKA is the major effector of cAMP. We further find that different bZip transcription factors are required for distinct aspects of the regenerative response, suggesting calcium/cAMP acts via multiple transcription factors. Our studies indicate that calcium and cAMP have conserved signaling roles and are rate limiting for regeneration. We are currenty exploring the relationship between calcium/cAMP signaling and other regeneration pathways.

Shipa R Ghosh et al. (2005) International Worm Meeting "Identifying novel components of muscle in C. elegans"

Ian A Hope, Shipa R Ghosh

[International Worm Meeting, 2005]

The smooth sinusoidal movements of C. elegans are effected by 95 obliquely striated bodywall muscle cells. There are also non-striated single-sarcomere muscles; the pharyngeal, intestinal, anal and sex-specific muscles. Force generated by the myofilament lattice of bodywall muscle is transmitted to the external cuticle through the attachment structures of the dense bodies, M-lines and fibrous organelles. Attachment structures are also present in the single-sarcomere muscle cells. Additional components of the lattice and associated attachment structures may be identified by investigating the subcellular localisation of endogenous proteins fused to GFP. The twin resources of the Promoterome and ORFeome are being used to construct Promoter::ORF::GFP fusions in a high-throughput manner using Multisite Gateway recombinational cloning. Transgenic lines are generated using these constructs to study the expression patterns of ORF::GFP fusion proteins in vivo. The genes selected for inclusion in this project were chosen because their respective promoters had been shown previously to drive expression of reporter genes in muscle cells. Including ORFs within Promoter::ORF::GFP constructs may reveal the subcellular localisation of the native protein. The genes W05F2.4, F07C3.4 and F02A9.3 were used to establish procedures and expression patterns will be presented. Relative localisation of the fusion proteins with well-known components of muscle structures will be investigated using reporter genes of differing colours. This would also facilitate visualisation of the various components of the contractile apparatus and associated attachment structures assembling throughout development. After initially identifying novel components, techniques such as RNAi and yeast two-hybrid experiments may be employed to clarify the roles and interactions of these proteins.

Ghosh, D. Dipon et al. (2013) International Worm Meeting "Neuropeptide modulation of C. elegans light avoidance circuitry."

Koelle, Michael R., Ghosh, D. Dipon, Nitabach, Michael N.

[International Worm Meeting, 2013]

Neuropeptides modulate neuronal circuit activity to regulate animal behavior. We used Caenorhabditis elegans as a model system to investigate the mechanism by which these neuromodulators fine tune cellular circuit outputs to optimize behavioral responses. Recently, homologs of an arthropod peptide Pigment Dispersing Factor (PDF) and its cognate receptor, PDF Receptor (PDFR), were identified in C. elegans. Secretion of PDF and its mammalian relative, Vasoactive Intestinal Peptide (VIP), is triggered by activation of light sensors and can modulate locomotor rhythms in insects and mammals, respectively. We found that null mutant worms lacking a PDF homolog exhibit aberrant light avoidance responses. Further analysis of these deficits indicates that this peptide could function in sensorimotor circuitry to modulate navigation in the presence of light. Our results suggest a link between circadian light entrainment in mammals and photophobic responses in the simpler worm, and a physiological mechanism by which neuromodulators regulate behaviors.

Rajarshi Ghosh et al. (2003) International Worm Meeting "Molecular genetics of male leaving, a sexually dimorphic behavior in C.elegans."

Jonathan O Lipton, Rajarshi Ghosh, Scott W Emmons

[International Worm Meeting, 2003]

In contrast to hermaphrodites, single C.elegans males when cultured on a small bacterial lawn do not remain on the food source indefinitely. Eventually an isolated male wanders off the food source. We refer to this sexually dimorphic behavior as leaving. We have characterized leaving and found that its properties are consistent with that of a spontaneous, goal-directed and regulated mate searching behavior. Leaving behavior is only expressed by adult males and its rate is governed by internal factors like nutritional status as well as external factors like presence of other males or hermaphrodites. We have developed a simple assay to quantitate this leaving behavior. We found that leaving is a stochastic process definable by a decay constant PL , which is the probability of leaving per hour. Leaving behavior, at least in part, should be dependent on locomotion. So the differences in locomotion of different mutants used in our study may be enough to account for the differences in PL and this might also account for the sexual dimorphism since males are more active than hermaphrodites. To address this issue , we used the leaving assay and demonstrated that there was apparently no correlation between activity (as determined by body bends per 20 seconds) and the probability of leaving in various mutant backgrounds. These observations support the hypothesis that leaving is not a secondary consequence of locomotion but is a specific regulated sexual behavior. The properties of leaving and the availability of a simple quantitative assay prompted us to investigate the genetic basis of this leaving behavior. We carried out a genetic screen for mutants with decreased PL values. So far we have isolated several las (leaving assay defective ) mutants. One of these mutants is an allele of the C.elegans serotonin reuptake transporter (CeSERT) gene, mod-5, which functions in serotonergic neurotransmission to clear the synaptic cleft of released serotonin. This suggests that modulation of serotonin and/or components of the serotonin pathway may play a critical role in leaving behavior. The finding is of particular interest because blocking the serotonin reuptake transporter in humans (with fluoxetine antidepressants) results in decrease in sex drive. It is interesting to note that of the three previously identified mod-5 alleles one of them has a relatively low PL value. We are currently investigating the genetic complexity at this locus that may give rise to such a result.

Rajarshi Ghosh et al. (2004) East Coast Worm Meeting "Serotonin and octopamine modulate thrashing behavior of C elegans"

Rajarshi Ghosh, Scott W Emmons

[East Coast Worm Meeting, 2004]

Certain mutants defective in male leaving behavior (these males, unlike wild type ones, do not leave a patch of food over a period of time) were also serotonin hypersensitive i.e. they got paralyzed in a 10mM solution of serotonin in M9 buffer. While studying the serotonin hypersensitivity at this concentration we found that ~70% of wild type worms were in a paralyzed state after 15 minutes of thrashing while 50% were in a paralyzed state after 20 minutes, implying that 20% of worms came out of paralysis state. We hypothesized that in liquid the worms might be going into a resting state and eventually coming out of it, and that this pattern is modulated by serotonin. To investigate such a phenomenon we studied thrashing in greater details. We found that in contrast to locomotion in agar plates, the body bends/20 seconds of wild type worms is ~ 4 times greater in M9. This is consistent with a previous observation that the worms exhibited a forward bias in their movement i.e. waves passed backwards ~4 times more frequently in M9 than when worms were moving on agar plates (Croll, 1975). Most interestingly, we observed that this thrashing pattern was interrupted by bouts of rest periods (~6-7 minutes), which for the wild type worm occurred after a period of ~ 50 minutes of thrashing from the time they were put in M9. The worms then come out of the resting state and continued thrashing. Given the striking differences in locomotion we wondered what the neural correlate of transition from slow (on plates) to fast (in M9) movement might be and what genes might be involved in such a process? In addition we also wanted to address the question of how such a behavior comes to a halt and then resumes again? To answer these questions we devised an assay where single worms were put into micro titer wells containing 200microliters of M9 buffer and watched every minute for 60 minutes and their thrash/rest pattern were recorded. We noted several interesting properties of thrashing. We found that the rearing temperature has no obvious detectable influence on the thrash/rest cycle but the assay temperature is critical for the wild type thrash/rest pattern. By using sensory pathway mutants like che-2(e1033) , osm-5(p118) and unc-86(sm117) we found that sensory input is essential for exhibiting wild type thrashing behavior. These mutants show very slow thrashing and the wild-type thrash-rest pattern is remarkably disrupted. We also found that the wild type pattern of thrash/rest cycle is disrupted by exogenous serotonin and octopamine. Wild type worms go more frequently into paralysis with increasing concentration of exogenous serotonin. Octopamine has the opposite effect i.e. it increases the thrash periods but has a moderate effect on paralysis periods. We found that like the null allele, a novel, mis-sense allele of mod-5 (the serotonin reuptake transporter in C.elegans.) exhibits defective thrashing behavior. Interestingly the thrashing defect of the mis-sense mutation can be supressed by exogenous octopamine. We are characterizing the genetic complexity of mod-5 locus with respect to thrashing behavior. Using mutants in the serotonergic pathway we also found that serotonin is required for wild-type thrash- rest cycle and excess serotonin tend to disrupt this pattern. We are characterizing the modulation of thrashing behavior by serotonin and octopamine in greater details.

Rajarshi Ghosh et al. (2008) C.elegans Neuronal Development Meeting "Individual differences in thermosensation and thermal avoidance behavior in C.elegans"

Leonid Kruglyak, Rajarshi Ghosh, William Ryu

[C.elegans Neuronal Development Meeting, 2008]

The behavioral output of an organism can be thought of as a temporally integrated series of ''sub-behaviors''. Individuals within and closely related species often exhibit heritable differences in sub-steps of a behavioral output. What genetic architecture and changes allows for such plasticity? We use responses to step increases in temperature by the nematode C.elegans to address this question. We devised an assay where the local temperature around a worm was raised in steps of 0.3, 0.8, 3 or 7.5 C for 0.5 seconds and its speed was monitored for 15 seconds at 28 frames per second after the initiation of the stimulus. The stereotyped behavior of the worms in response to such stimuli allowed us to generate a panel of phenotypes including maximum speed, latency to response and duration of reversals after the initiation of the stimulus. Comparison of Bristol and the Hawaii isolates revealed intriguing differences in response to these stimuli. We also found striking sexually dimorphic response in Bristol strain. Males coil in response to a 7.5C step. This response was absent in Hawaii strain. We are testing 239 advanced intercross recombinant inbred lines (RILs) from reciprocal crosses between Bristol and Hawaii strains to study the pattern of segregation of these phenotypes in both sexes. In addition to the RILs we studied effects of mutation in several candidate genes. Our preliminary analysis suggests that responses to above stimuli are encoded by combination of distinct neurotransmitter systems. Glutamate and neuropeptides are required for responses to 0.3 C and 0.8 C rise in temperature whereas neuropeptides mediate responses to a temperature rise of 7.5 C. We found that ttx-1 and ttx-3 mutants, which have impaired thermotaxis, abolish responses to 0.3 C-step but retain Bristol-like responses at other stimuli. Responses to all of these stimuli are abolished when command neurons essential for backward but not forward movement are genetically ablated.The pattern of variation of the responsesamong RILs along with the candidate genes may allow us to decipher how animals encode different step increases in temperature.