Tanaka, Ryusei et al. (2021) International Worm Meeting "Reproductive incompatibility among populations of Caenorhabditis inopinata"

Kikuchi, Taisei, Sugimoto, Asako, Yoshida, Akemi, Tanaka, Ryusei

[International Worm Meeting, 2021]

Caenorhabditis inopinata, the sibling species of C. elegans, recently discovered in Ryukyu islands Japan, has a distinct lifestyle from other known Caenorhabditis species. The gonochoristic species inhabits in syconia of a fig tree (Ficus septica) and uses the pollinator wasp (Ceratosolen sp.) as a vector to move from old to new syconia. We isolated C. inopinata from fig syconia collected in seven Ryukyu islands and Taiwan (distance in 730 km), and established over 20 single female lines. Phylogenetic analyses of those lines revealed that C. inopinata can be separated largely into two geographical groups (Okinawa and Ishigaki groups). Although we couldn't identify clear morphological differences, the genetic distance of the two groups calculated by genome comparisons was relatively large as an intra-species diversity. We therefore sought to clarify mating and reproductive compatibilities between the two geographical groups. Using three C. inopinata lines each from Okinawa and Ishigaki groups, respectively, we observed mating behaviours, embryo developments and reproductions for all line combinations and directions (male - female). Mating behaviours were observed in all combinations though sexual attraction levels seem lower in inter-group than intra-group combinations. However, offspring (F1) numbers were smaller in inter-group combination, especially incomplete embryo developments and almost no offspring were observed at Okinawa male and Ishigaki female combination. These results suggest that speciation in C. inopinata has possibly been accelerated by the characteristic lifestyle and the island effect in this small area.

Hunt, Vicky L. et al. (2019) International Worm Meeting "Loss of the ERGO-1 small RNA pathway in Caenorhabditis inopinata."

Hunt, Vicky L., Murase, Kazunori, Yoshida, Akemi, Kikuchi, Taisei, Tanaka, Ryusei

[International Worm Meeting, 2019]

The ERGO-1 small RNA (sRNA) pathway is best characterised in C. elegans where it is involved in regulating the expression of tandemly duplicated genes and pseudogenes. We are investigating sRNAs in C. inopinata, the closest known relative of C. elegans. Many of the sRNA pathways and their associated sRNAs are conserved between these two species. However, orthologues of the genes coding for the ERGO-1 Argonaute protein and other genes involved in the ERGO-1 pathway have been lost from C. inopinata. This loss is likely to be the result of high levels of transposase activity in the C. inopinata genome. The 26G small interfering RNAs associated with the ERGO-1 pathway are also absent in C. inopinata and the sRNA profiles in these nematodes are similar to C. elegans ergo-1 mutants. To understand the conservation and diversification of the ERGO-1 pathway in nematodes more widely we have identified ERGO-1 pathway orthologues across diverse nematode species. We find that although the ergo-1 Argonaute gene is well conserved amongst nematodes, other genes involved in this pathway are absent from nematodes outside of the Caenorhabiditis clade. We hypothesise that the ERGO-1 pathway found in C. elegans is unique to this clade of nematodes but has been lost in C. inopinata.

Sun, Simo et al. (2021) International Worm Meeting "The smallest genome in the genus Caenorhabditis"

Sun, Simo, Kanzaki, Natsumi, Yoshida, Akemi, Kikuchi, Taisei, Maeda, Yasunobu, Mehmet, Dayi

[International Worm Meeting, 2021]

Caenorhabditis elegans is a powerful laboratory model that has provided several key findings in molecular and developmental biology and neuroscience in the past decades. However, only little is known about the evolutionary history of the nematode and the relatives. Recent extensive surveys of new Caenorhabditis species around the world revealed that the diversity in the genus is bigger than we previously expected. Those resources are useful to get evolutionary insights for better understanding of biological phenomena identified in C. elegans researches and provide opportunities to perform deep evolutionary analyses on morphology, behaviors and genomes. Here we report a new Caenorhabditis species C. sp. 36, which has the smallest genome in the genus. The new gonochoristic species was isolated from a weevil (Niphades variegatus) collected in the dead log of Masson's pine in Tokyo Japan. Morphologically, the species possesses the typical characteristics of the Elegans supergroup species except the body size is a little smaller. Using Illumina, Nanopore and Hi-C technologies, we assembled the C. sp. 36 genome into six big scaffolds accounting for the chromosomes. The genome assembly size was as small as ~58Mb, the smallest among the well-defined Caenorhabditis genomes. Phylogenetic analysis revealed that C. sp. 36 is a close relative of C. japonica whose genome size is one of the biggest in the genus (156 Mb). For a comprehensive genome comparison with C. sp. 36, we also sequenced C. japonica genome using aforementioned technologies and achieved a big improvement from the wormbase ver WS279. Though the two genome sizes are different by three times, similar numbers of protein coding genes (16929 and 17652 genes, respectively) were predicted for C. sp. 36 and C. japonica, which are comparable numbers with other Caenorhabditis species. Whereas a total CDS span dose not differ much from other spices, intron and intergenic regions showed big size differences. Compared to C. elegans, C. sp. 36 has ~19.5Mb and ~18.2Mb smaller intron and intergenic spans, respectively. In contrast, those of C. japonica are ~26.3Mb and ~32.8Mb larger than of C. elegans, respectively. A deeper intron analysis revealed that although intron birth/death trends differed depending on each lineage of Caenorhabditis, each-intron length rather than per-gene intron counts mainly contribute to the intron span differences. Repeat analyses showed that transposons, especially DNA transposons are the main factors involved in the intergenic region differences.

Yoshida K et al. (2024) STAR Protoc "Protocol for forward genetic screening to identify novel factors involved in a biological process in Caenorhabditis elegans."

Yoshida K, Suehiro Y, Mitani S

[STAR Protoc, 2024]

Forward genetic screens have been a powerful tool for discovering genes involved in various biological processes in Caenorhabditis elegans. Here, we present a protocol for forward genetic screening to identify novel factors involved in a biological process in C.elegans. We describe steps for mutagenesis, screening, and backcrossing. To save time and effort, we also detail procedures for utilizing whole-genome sequencing to exclude mutants of previously characterized genes from crosses for mapping mutations. For complete details on the use and execution of this protocol, please refer to Yoshida et al.¹.

Kikuchi, Taisei et al. (2021) International Worm Meeting "An alternative ERGO-1 pathway in a sibling species of C. elegans, C. inopinata"

Sugimoto, Asako, Kikuchi, Taisei, Tanaka, Ryusei, Yoshida, Akemi, Sun, Simo, Hunt, Vicky

[International Worm Meeting, 2021]

The recent discovery of Caenorhabditis inopinata, a sibling species of C. elegans, provides an opportunity to investigate the conservation and diversification of small RNA (sRNA) pathways in two closely related species. We report that the microRNA, PIWI and male-expressed ALG-3/4 siRNA pathways are highly conserved in C. inopinata and C. elegans. However, C. inopinata has lost key components of the female-expressed ERGO-1 siRNA pathway associated with the clearance of transcripts. C. inopinata do not express the ERGO-1 associated 26G primary siRNAs and have similar sRNA expression profiles to C. elegans ergo-1 and eri-6 mutants. However, expression of the downstream secondary 22G siRNAs is retained in C. inopinata. We have identified a class of 22U siRNAs that are candidates for an alternative ERGO-1 siRNA. The 22U loci overlap with 22Gs and target similar genes but, unlike C. elegans 26Gs, are not dicer-processed. We also identified an Argonaute protein with an expression profile similar to the C. elegans ERGO-1, as a candidate for an alternative ERGO-1 Argonaute protein. Our results indicate that there has been a partial loss of the ERGO-1 pathway in C. inopinata and an alternative ERGO-1 pathway has evolved.

Kounosu, Asuka et al. (2021) International Worm Meeting "Chromosome dynamics in sex determination of the parthenogenetic nematode Strongyloides ratti"

Sugimoto, Asako, Maruyama, Haruhiko, Murase, Kazunori, Yoshida, Akemi, Hunt, Vicky, Sun, Simo, Kikuchi, Taisei, Kounosu, Asuka

[International Worm Meeting, 2021]

The genus Strongyloides is unique among nematodes in having alternate free-living and parasitic generations. The parasitic female (PF) inhabits the small intestine of its mammal host and produces clonal offspring by mitotic parthenogenesis. Eggs produced by PF are excreted with the feaces and develop into either infective larvae (iL3) directly or free-living males and females. The free-living adults mate and produce eggs which develop into iL3. It was reported that Strongyloides ratti, a rat parasite, has three chromosomes and one of them is a sex chromosome, which determines their sex by XX/XO system. However, it remains unclear how mitotic parthenogenetic PF produces XX and XO eggs though it is presumed that the male karyotype (XO) is generated by missegregation of the sex chromosome like the C. elegans male generation. In this study we investigated details of the chromosome structure of each developmental stages of S. ratti using the single-worm sequencing technique and the multiplex colored FISH observation. In the single-worm analysis, we expected a half depth of mapping coverage in sex chromosome compared to autosomes in free-living males (XO). However, the sex-chromosome of the male exhibited ~1/3 depth of the autosomes. Furthermore PF and free-living female (XX) showed lower coverage depth of the sex-chromosome and the auto:sex ration varied from 1:0.8 to 1:0.5. Additionally, in germline depleted PF, sex-chromosome showed almost equal coverages as autosomes. These results indicate that chromosome manipulations occurs in the PF germline to produce free-living males and the S. ratti sex determination system is not a simple XX/XO. Multiplex colored FISH analyses, recently developed for C. elegans in-detail chromosome observation, provided visual clues to understanding the dynamics of S. ratti chromosomal structure and the puzzling sex-determination system of Strongyloides.

Miyanishi, Yosuke et al. (2011) International Worm Meeting "Functional imaging of neuronal activity for 2-nonanone stimulation."

Nakai, Junichi, Kimura, Kotaro, Miyanishi, Yosuke

[International Worm Meeting, 2011]

To better understand the neural basis that regulates a worm's sensory behavior and its modulation by learning, we are studying avoidance behavioral responses to 2-nonanone. We previously reported that the avoidance behavior to 2-nonanone is enhanced, rather than reduced, after preexposure to the odor, and this enhancement is acquired as a non-associative dopamine-dependent learning (Kimura et al., J. Neurosci., 2010; Fujita and Kimura, this abstract). In addition, we observed that worms respond to a spatial gradient of 2-nonanone (Yamazoe and Kimura, CE Neuro, 2010), which cannot be simply explained by the pirouette or weathervane strategies. 2-nonanone is mainly sensed by the AWB neurons, which have been shown to exhibit odor-OFF response in aqueous step stimulation with 2-nonanone (Troemel et al., Cell 1997; Ha et al., Neuron 2010). To understand how the neuronal circuits of worms regulate the characteristic 2-nonanone behavioral response, we are monitoring calcium changes in the AWB and downstream neurons using G-CaMP 4 (Shindo et al., PLoS ONE, 2010). We thank Drs. S. Oda, K. Yoshida, and Y. Iino (U. Tokyo) for suggestions on microfluidics; M. Hendricks and Y. Zhang (Harvard) for aqueous 2-nonanone stimulation; and E. Busch and M. de Bono (MRC) for gaseous microfluidic stimulation.

C Leopold Kurz et al. (2002) European Worm Meeting "Inducible innate immune defences in C. elegans; a TGF connection."

Gustavo Mallo, C Leopold Kurz, Samuel Granjeaud, Yuji KOHARA, Jonathan Ewbank

[European Worm Meeting, 2002]

C. elegans is emerging as a very promising model for the study of host-pathogen interactions (Aballay and Ausubel 2002; Ewbank 2002). In addition to its utilisation for the identification of bacterial virulence factors, it can be used to investigate conserved aspects of innate immunity. Both plants and vertebrates respond to infection by the production of antimicrobial proteins and peptides. To determine whether C. elegans possesses an analogous inducible system of defence, we used high-density cDNA arrays to look for genes that are induced upon infection with the Gram-negative bacterium Serratia marcescens. We found that several hundred genes show significant alterations in their level of expression following infection. Among the most robustly induced are genes encoding lectins and lysozymes, known to be involved in immune responses in other organisms. Inactivation of the lysozyme gene lys-1 by RNAi renders worms more susceptible to infection. Conversely, its over-expression augments the resistance of worms to S. marcescens. Certain of the infection-inducible genes are under the control of the dbl-1/TGFb pathway, and it had previously been shown that their expression was abolished in dbl-1 mutants (Mochii, Yoshida et al. 1999). Consistent with this, dbl-1 mutants exhibit increased susceptibility to infection. We therefore conclude that C. elegans does possess an inducible system of antibacterial defence, that in part rests on the TGFb signalling pathway. In our future work, we aim to determine the specificity of the response and the degree to which the underlying mechanisms have been conserved in other species.

McCormick K et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Going with the Flow: A Microfluidic Approach to the Behavioral Analysis of Chemotaxis in C. elegans"

McCormick K, Lockery S R, Sottile M

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

C. elegans orients to chemosensory gradients using two main strategies. In klinokinesis (also known as the pirouette strategy) the worm randomly chooses a new heading upon sensing a decrease in concentration of a favored chemical. In klinotaxis (also known as the weathervane strategy) the worm directs the course of its forward movement such that its path curves toward increasing concentrations of a favored chemical. Whereas klinokinesis has been a subject of experimental study for some years, klinotaxis has only recently been characterized. In an initial report, Iino & Yoshida find that the curving rate of the worm increases as a linear function of the gradient steepness in the direction perpendicular to the worm's heading (hereafter, the transverse direction) [1]. This relationship suggests that the head swings of the worm's undulatory locomotion may be involved in sensing the gradient and executing gradual curves during klinotaxis. We have developed a microfluidic device that has two key advantages over studying klinotaxis in freely moving worms. First, the concentration difference during head swings is known precisely because it is under the control of the experimenter rather than the animal. This should make it possible to measure behavioral thresholds more easily. Second, the concentration gradient in the transverse direction can be manipulated independently of the concentration gradient in the longitudinal direction. This allows us attribute changes in head sweep behavior specifically to the transverse gradient. In the microfluidic chip, we find that worms bias their mean head swing angle toward higher attractant concentrations. Furthermore, head-swing bias is proportional to the difference in concentration between the dorsal and ventral sides of the worm. These findings are consistent with the hypothesis that path curvature towards chemosensory peaks is implemented through bias to the head swings of undulatory locomotion. We are currently addressing the contribution of candidate sensory and motor neurons to klinotaxis behavior in the microfluidic chip using cell ablation, calcium imaging, and photoactivation experiments.

Kerri Spilker et al. (2007) International Worm Meeting "Synaptic vesicle localization is affected by in a new mutation in mec-7/b-tubulin."

Kerri Spilker, Kang Shen

[International Worm Meeting, 2007]

Synapse formation in C. elegans relies on the transport of synaptic proteins and synaptic vesicles to specific locations in the cell via microtubules and motors. Once at the proper location, the synapse is assembled and synaptic vesicles are clustered. We are interested in how synaptic specificity is achieved in the C. elegans nervous system. To study this, we constructed a transgenic line with a GFP cytoplasmic marker and mCherry-tagged RAB-3, a synaptic vesicle marker, and looked for mutations that affect neuronal morphology and synaptic vesicle localization. In our screen we identified a new mutation in the coding region of the touch cell-specific <font face=symbol>b</font>-tubulin, mec-7(wy116). Previous studies have shown that mec-7 is expressed primarily in the six touch neurons of C. elegans and is required for sensing light touch (Chalfie, M. and J. Sulston, 1981. Development 82:358-370; Hamelin et al., 1992. The EMBO Journal 11(8):2885-2893) Our new mec-7 mutation leads to a loss of synaptic vesicle accumulation at PLM synaptic sites in the ventral nerve cord and synaptic vesicles are visible at ectopic locations along the lateral axon of PLM. Localization of the synaptic proteins SNB-1 and GIT-1 is also defective in our mutant, but neuronal morphology is wild-type. mec-7(wy116) is mildly Mec, but other alleles of mec-7 (e1506, e1527) do not phenocopy the synaptic vesicle localization defect. Our new allele is a missense mutation that alters a highly conserved Thr at position 409 to Ile. Crystal structures of tubulin indicate that this residue is on the face of tubulin that interacts with kinesin motor proteins (Nogales et al., 1998. Nature 391:199-203). Because we see synaptic vesicles along the lateral axon of PLM, we believe that kinesin-mediated vesicle transport does occur in these mutants. We will explore further how mec-7(wy116) causes a specific, subcellular defect in vesicle trafficking and localization. We hypothesize that Thr-409 is required for targeting kinesin cargo to subcellular compartments of the neuron. In vitro data suggests that this residue of tubulin is phosphorylated and we are exploring this possibility (Yoshida et al., 2003 Eur J Biochem 270: 1154-1163).