Lightfoot, James et al. (2015) International Worm Meeting "Self-Recognition Prevents Cannibalism in Predatory Nematodes."

Lightfoot, James, Moreno, Eduardo, Susoy, Vladislav, Sommer, Ralf, Wilecki, Martin, Rodelsperger, Christian

[International Worm Meeting, 2015]

Self-recognition described as the capacity to discriminate between identical and foreign tissue is observed abundantly throughout nature and is exploited for a plethora of diverse biological functions. These range from the adaptive immune response in jawed vertebrates to neuronal wiring and dendrite self-avoidance in fruit flies to inducing swarming behaviour in bacteria. However, although self-recognition is observed ubiquitously in nature, relatively little is known about the mechanisms behind this phenomenon and surprisingly, despite the pre-eminence of Caernorhabditis elegans as a model organism, evidence of self-recognition has thus far never been described in nematodes. Our recent investigations explored the feeding dynamics in the predatory nematode Pristionchus pacificus and revealed P. pacificus to be voracious killers of C. elegans. We have subsequently, analysed predatory interactions in the context of self-recognition by investigating predation between P. pacificus and its self-progeny. Predation occurs interspecifically between a wide range of closely related nematode species however never on self-progeny, a mechanism that is conserved amongst other Pristionchus species. Furthermore, the ability to determine self from non-self is highly specific with even closely related strains of P. pacificus perceived as prey, while self-progeny are ignored. The identification of self-progeny is dependent on interactions with surface bound molecules, a process which is maintained despite severe starvation and additionally, not disrupted in a host of cuticle morphology mutants. Finally as even closely related P. pacificus strains predate one another while distinguishing and ignoring self-progeny, we have exploited single nucleotide polymorphisms between two strains and generated recombinant inbred lines (RILs) to isolate the genetic component of self-recognition. Each of the RILs was successfully predated by only a single parental lineage facilitating quantitative trait locus (QTL) mapping resulting in the identification of a putative candidate gene and the underlying molecular mechanism. We thus present the first evidence of self-recognition in nematodes, a phenomenon that enables P. pacificus to avoid cannibalism while also promoting the killing of larvae from potential competing nematodes.

Lightfoot, James W. et al. (2013) International Worm Meeting "The Role of Pharyngeal Glands in Nematode Feeding and Diet."

Lightfoot, James W., Sommer, Ralf J.

[International Worm Meeting, 2013]

Nematode pharyngeal glands have historically been associated with feeding as well as cuticle moulting, although little is understood of the molecular biology underlying these processes. By utilising two evolutionary divergent nematode species with differing diets and feeding traits we are elucidating the roles of the pharyngeal gland cells and their function in feeding, diet and other cellular processes. The model organisms C. elegans and P. pacificus provide an apt system with which to analyse the pharyngeal glands, as while both nematode species contain several pharyngeal gland cells, C. elegans is predominantly a bacterial feeder, whereas P. pacificus is capable of predating other nematode species in addition to its bacterial feeding habits. Accordingly, gland cells display an evolutionary divergence in both quantity and morphology between species. The pharynx of C. elegans contains five pharyngeal gland cells, a large dorsal gland (g1D) exiting at the mouth opening, two ventral glands (g1V) emptying into the median bulb and two ventral glands (g2) exiting into the terminal bulb. The pharynx of P. pacificus contains three gland cells, lacking the g2 glands, and additionally the g1D is morphologically distinct as the gland duct exits through the predatory dorsal tooth used to eviscerate other nematodes before feeding. Furthermore, substantial divergence in the function of pharyngeal glands between these two nematode species has been predicted. The dorsal gland in the bacterial feeding nematode C. elegans is stimulated through inputs from the isthmus while the dorsal gland in the bacterial and predatory feeder, P. pacificus receives inputs from the corpus. This dramatic shift in regulation may indicate an alteration in gland cell regulation towards additional predatory functions. Cell ablation experiments removing g1D cells however reveal much functional conservation as animals become starved due to a bacterial plug blocking the pharynx in both species. Therefore, we have begun generating the transcriptome of the pharyngeal gland cells using mRNA tagging followed by RNA-seq with which we will pursue the genetic candidates behind gland cell function and the expanded diet evident in P. pacificus..

Lightfoot, James (2021) International Worm Meeting "A genetically linked gene pair determines organismal self-identity in predatory nematodes"

Lightfoot, James

[International Worm Meeting, 2021]

The ability to distinguish self and kin from foreign entities has been observed across the natural world and is associated with an immensely diverse spectrum of biological processes, including both cooperative and competitive behaviours. While behaviours associated with the identification of self and kin are commonplace, understanding the proximate mechanisms are challenging due to a lack of genetic, molecular and neurobiological techniques available in many of these organisms. Nematodes are a group of organisms without such limitations and have been used as a mainstay of organismal behavioural research. Further, the recently discovered self-recognition behaviours in the free-living nematode Pristionchus pacificus provides an ideal system to uncover the mechanism involved in unprecedented detail. P. pacificus are omnivorous nematodes capable of supplementing their bacterial diet by predating on the larvae of other nematodes. They also display cannibalistic behaviours towards conspecifics but do not cannibalise their own self-progeny. This self-recognition system is dependent on the small-peptide SELF-1, which contains a hypervariable region and is expressed in all hypodermal cells. While, self-1 is undoubtably an important component in the self-signal, recent experiments exploring ecologically relevant con-specific interactions demonstrated it is not the only factor involved. To identify additional self-recognition elements, we explored genes closely linked to self-1, as self-recognition components are often clustered in small genetic intervals. Two genes were prioritised due to being lineage specific orphan genes like self-1. CRISPR/Cas9 induced mutations in either gene candidate displayed no self-recognition defect. However, a double mutant between self-1 and one gene candidate displayed a self-recognition defect greater than self-1 alone. This gene was named self-2 and is the second gene in a two gene operon located adjacent to self-1. Subsequent analysis of self-2 revealed it consists of 249 amino acids with a signal peptide and transmembrane domain. Ongoing experiments seek to clarify any interaction between self-1 and self-2 as well as any involvement from the additional operon gene. Furthermore, we are utilising surface-sensitive analytical mass spectrometry to determine any influence these mutants may have on cuticle surface coat composition and its effect on the self-recognition system.

Butler JA et al. (1994) Worm Breeder's Gazette "Looking For Extracellular Matrix Proteinases in C. elegans"

Kramer JM, Butler JA

[Worm Breeder's Gazette, 1994]

LOOKING FOR EXTRACELLULAR MATRIX PROTEINASES IN C. ELEGANS. James A. Butler and James M. Kramer, Northwestern University Medical School. Department of CMS Biology, Chicago IL 60611

Wissmann AK et al. (1994) Worm Breeder's Gazette "Characterization of the let-502 Gene"

Mains PE, Wissmann AK, McGhee JD

[Worm Breeder's Gazette, 1994]

Characterization of the let-502 gene Andreas Wissmann, James D. McGhee and Paul E. Mains, Dept. of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada T2N 4N1

Martinez-Perez, Fadri et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Cohesin loading by SCC-2 is required for meiotic chromosome morphogenesis and for activation of the pachytene DNA damage checkpoint"

Testori, Sarah, Martinez-Perez, Fadri, Lightfoot, James

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The sister chromatid cohesion complex is required for the formation of a physical linkage between the two copies of a chromosome that are created during DNA replication, and the establishment and ordered dissolution of cohesion are essential for correct chromosome segregation. Loading of cohesin on chromosomes starts before S phase and requires a protein complex containing SCC-2 and SCC-4. During a screen for meiotic mutants we isolated an allele of scc-2, scc-2 (fq1), carrying a premature stop codon. Although scc-2 (fq1) homozygous mutants are viable, their germlines display abnormal chromosome morphology throughout meiotic prophase and immunostaining experiments reveal that cohesin subunits and synaptonemal complex components fail to associat e with meiotic chromosomes. Despite this, RAD-51 staining shows that meiotic recombination is initiated with normal dynamics in scc-2 (fq1) mutants, in a SPO-11-dependent fashion, but that recombination intermediates accumulate extensively and persist until diplotene. Two lines of evidence suggest that these recombination intermediates remain unrepaired: first, chromosome fragments are observed in scc-2 (fq1) oocytes and second, oocytes of scc-2 brc-2 double mutants do not show the chromosomal aggregates seen in brc-2 oocytes, which result from the illegitimate repair of DNA double strand breaks (DSBs). Surprisingly, this apparent inability to repair DSBs does not activate the pachytene DNA damage checkpoint, since scc-2 (fq1) mutants do not display increased levels of apoptosis. In agreement with a failure to activate the DNA damage checkpoint we show that phosphorylated targets of ATM and ATR kinases do not localize to meiotic chromosomes in scc-2(fq1) mutants. Furthermore , spo-11 scc-2 double mutants show a delay in the formation of RAD-51 filaments following gamma irradiation. These results suggest that cohesins are required for the efficient processing of DSBs and to signal the presence of DNA damage during meiosis.

Wilecki, Martin et al. (2013) International Worm Meeting "The monoamine neurotransmitter serotonin shows evolutionarily divergent effects on feeding behavior in Pristionchus pacificus."

Lightfoot, James W, Sommer, Ralf J, Wilecki, Martin

[International Worm Meeting, 2013]

The ecology and behavior of the diplogastrid nematode Pristionchus pacificus differs substantially from C. elegans. While both nematodes can be cultured on bacteria, P. pacificus is able to exploit alternative food sources. Tooth-like denticles in the mouth enable it to feed on other nematodes and fungi. We are interested in the neurobiological basis behind the predatory vs. bacterial feeding in P. pacificus. Monoamine neurotransmitters regulate several aspects of feeding behavior in C. elegans. Although antibody stainings reveal similar expression patterns in both nematodes, the response to these neurotransmitters in pharmacological experiments differ substantially. Most tested neurotransmitters elicit opposite responses in pharyngeal pumping assays. For example, serotonin is known to stimulate pharyngeal pumping in C. elegans, but it dramatically decreases pharyngeal pumping in P. pacificus. Serotonin also triggers movement of the claw-like dorsal tooth in P. pacificus, which makes it an interesting candidate for more detailed studies of the neurobiology of the predatory feeding. The rate-limiting serotonin synthesis enzyme tryptophan hydroxylase (TPH-1) and the serotonin transporter MOD-5 are highly conserved between P. pacificus and C. elegans. We are using reporter constructs to visualize expression of the underlying genes and use cell ablation experiments to elucidate key neurons involved in regulating the complex feeding behaviors in P. pacificus. We also plan to generate mutants of tph-1 and mod-5 using TALENs for phenotypic analysis of their effects on feeding behaviors.

Eren, Guniz Goze et al. MicroPubl Biol

Eren, Guniz Goze, Roca, Marianne, Han, Ziduan, Lightfoot, James W

[MicroPubl Biol, 2022]

Transgenes are widely used throughout molecular biology for numerous applications. In Caenorhabditis elegans, stable transgenes are usually generated by microinjection into the germline establishing extrachromosomal arrays. Furthermore, numerous technologies exist to integrate transgenes into the C. elegans genome. In the nematode Pristionchus pacificus, transgenes are possible, however, their establishment is less efficient and dependent on the formation of complex arrays containing the transgene of interest and host carrier DNA. Additionally, genomic integration has only been reported via biolistic methods. Here we describe a simple technique using UV irradiation to facilitate the integration of transgenes into the P. pacificus genome.

Okumura, Misako et al. (2015) International Worm Meeting "Serotonin regulates dynamic feeding mode switching inducing predatory kinetics in Pristionchus pacificus."

Sommer, Ralf J., Lightfoot, James W., Okumura, Misako, Wilecki, Martin

[International Worm Meeting, 2015]

Pristionchus pacificus and its relatives are omnivores and are capable of consuming both bacteria and also predating other nematodes including the larvae of Caernorhabditis elegans. To this end Pristionchus species show specific predatory adaptations in the form of teeth-like denticles for lacerating open the cuticle of their prey. Accompanying these teeth structures, the variation in diet induces distinct feeding modes with predation characterised by slowed pharyngeal pumping rates and stimulation of tooth activity, while during bacterial feeding, pumping rates are higher and the tooth remains static. The switch between these distinct feeding modes is rapid, highly dynamic, and dependent on their immediate diet however little is known of the neurological bases regulating the feeding mode switch. Therefore, to elucidate the neurobiological basis underlying the regulation of the predatory feeding behaviour, we conducted pharmacological experiments analysing the effects of several neurotransmitters on pharyngeal pumping and tooth movement rate. Only the addition of serotonin recapitulated the feeding kinetics observed during predatory feeding indicating a previously uncharacterised role for this neurotransmitter. Immunostaining against serotonin revealed an additional serotonergic neuronal pair in the pharynx of P. pacificus compared to C. elegans. These likely correspond to P. pacificus NSM and the interneurons I1. We have subsequently identified the P. pacificus homologues of the serotonin synthesis enzyme (ppa-tph-1) and serotonin transporter (ppa-mod-5) for targeting by CRISPR/Cas9 in order to further investigate the neuronal networks regulating the behavioural switch and for understanding the evolution of these complex behaviours. .

Kulkarni, Arpita et al. (2015) International Worm Meeting "The highly divergent germ line in Strongyloididae."

Holz, Anja, Lightfoot, James W, Kulkarni, Arpita, Roedelsperger, Christian, Streit, Adrian, Harbecke, Dorothee

[International Worm Meeting, 2015]

Parasitic nematodes of the genus Strongyoides are small intestinal parasites of various vertebrates including man. Strongyloides spp. alternate between parthenogenetic parasitic and facultative free-living sexual generations. Generally, all progeny of free-living parents is female and parasitic. Whereas, Parastrongyloides trichosuri reproduces sexually, produces males and females in both generations and can form many consecutive free-living generations.Although superficially the gonads of free-living Strongyloides spp. appear rather similar to those of Caenorhabditis elegans, there exist dramatic differences between them. Most obvious is a population of non-dividing giant nuclei in the distal gonadal syncytium, which is populated by mitotically dividing germ line stem cells in C. elegans. These giant nuclei appear to be an evolutionary novelty restricted to the Strongyloididae and their close relatives (e.g. Rhabditophanes).Combining electron microscopy, light microscopy including immuno-staining for multiple histone modifications and FISH, bromo-dUTP incorporation experiments, and quantitative DNA and RNA sequencing we present a detailed characterization of the gonads of both sexes of free-living S. ratti, S. papillosus and Parastrongyloides trichosuri. The principle findings are:i) The distal portion of the gonad shows all signs of high gene expression activity hinting to nurse cell activity. In the giant nuclei of this region the DNA is highly amplified with autosomal regions present in higher copy numbers than X chromosomal regions. X linked genes are, on average, expressed at a lower level than autosomal ones suggesting that differential DNA amplification serves as a mechanism for the control of gene expression.ii) In S. ratti germ cells H3K9 tri-methylation and H3S10 phosphorylation appear mutually exclusive marking the autosomes and the X chromosome respectively.iii) New DNA synthesis is very low if not absent in gonads of adult free-living Strongyloides spp. suggesting that there is no mitotically dividing stem cell population within the adult germline.iv) As expected, genetically male determining sperm is present in P. trichosuri and absent in S. papillosus. Contrary to expectations, nullo-X sperm appears to exist in S. ratti indicating differential mechanisms to avoid the formation of males..