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[
International Worm Meeting,
2015]
In 2013, a novel nematode species was discovered in the fresh figs of Ficus septica in Okinawa. Subsequent DNA sequencing revealed that this species belongs to Caenorhabditis. Here, some biological characteristics of C. sp. 34 are described. C. sp. 34 is an exceptional member of Caenorhabditis in a number of respects. C. sp. 34 animals can grow to be nearly twice as long as C. elegans (1.5-2 mm), and take about twice as long to develop (~8 days at 20degC). This length difference in adults is largely due to post-embryonic events as C. sp. 34 embryos are about 19% longer than C. elegans embryos. C. sp. 34 sperm are enormous (about three times longer in diameter than C. elegans sperm), whereas the C. sp. 34 female tail spike is about half as long as that of the C. elegans hermaphrodite. However, examination of Hoechst-stained diakinesis oocytes reveals that, like C. elegans, C. sp. 34 has six chromosomes. No differences in the number of intestinal nuclei were observed between C. sp. 34 and C. elegans. Preliminary fluorescent microscopy observations suggest that the somatic nucleus number, hypodermal nucleus ploidy, and genome size of C. sp. 34 is comparable to that of other Caenorhabditis species. Additionally, mating tests show that C. sp. 34, C. sp. 35 (which is also fig-associated), and C. elegans are distinct biological species, and reproductive barriers include lack of sperm transfer, lack of fertilization, and embryonic inviability. This work has been concurrent with a larger collaborative effort, whose ongoing efforts include investigations into genomics, population genetics, and developmental biology. C. sp. 34 is an exciting species that will likely prove fruitful in future evolutionary studies.
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Maeda, Yasunobu, Kikuchi, Taisei, Sun, Simo, Yoshida, Akemi, Kanzaki, Natsumi, 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.
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[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2014]
Nematodes have many different reproductive strategies along with their divergent life-histories; the ability of hermaphrodite to self- and cross-fertilize is useful for genetic manipulation. Here we demonstrate the hermaphroditism of the fungal feeding nematode Bursaphelenchus okinawaensis, which was formerly described as a parthenogenetic nematode, and we show its other unique sexual characteristics. To determine the precise reproductive modes (i.e. parthenogenesis or hermaphroditism) in B. okinawaensis, we performed the following experiments: 1) observation of the pronuclear and chromosome behavior during oogenesis and early embryogenesis; 2) observation of the spermatogenesis during the L4 stage; 3) investigation of sperm utilization; 4) investigation of the phenotypic segregation after cross-mating using an EMS-induced visible mutant. The results of these experiments clearly showed the hermaphroditism of B. okinawaensis. We then investigated the mating preference and spermatid size between males and hermaphrodites. B. okinawaensis males successfully mated only with sperm-depleted old hermaphrodites and the spermatid sizes of males were almost the same as those of hermaphrodites. Moreover, the sex ratio of cross-fertilized progeny in B. okinawaensis was highly skewed toward hermaphrodites. To determine the sex distortion and determination mechanism, we then evaluated the activity of sperm from males and investigated the impact of various environments, autosomal sex determination genes and endosymbiotic microbes. The exact mechanism of the sex distortion and determination in B. okinawaensis is still unclear but seems to involve other unknown factors. Because the hermaphroditic nematode B. okinawaensis has an unique life history (i.e. fungal feeding, insect and plant association) and is phylogenetically distant from Caenorhabditis nematodes, it will be useful for future studies about the evolution of sex systems, feeding behaviors and parasitism.
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Kikuchi, Taisei, Hoshi, Yuki, Namai, Satoshi, Tsuyama, Kenji, Kumagai, Ryohei, Sugimoto, Asako, Kanzaki, Natsumi
[
International Worm Meeting,
2017]
Caenorhabditis sp. 34 is a sister species of C. elegans recently isolated from the syconia of the fig Ficus septica on Ishigaki Island, Japan (see abstract by T. Kikuchi, et al.). C. sp. 34 is gonochoric and shares typological key characters with other Elegans supergroup species, but strikingly, adults are nearly twice as long as C. elegans. The optimal culture temperature for C. sp. 34 is significantly higher (27 deg C) than that of C. elegans (20 deg C). Young adult males and females tend to form clumps, and Dauer larvae are rarely observed in laboratory culture conditions. Recently the C. sp. 34 genome assembly was produced into six chromosomes (see abstract by T. Kikuchi, et al.). The marked differences from C. elegans in morphology, behaviors and ecology, and the availability of the complete genome sequence make C. sp. 34 highly attractive for comparative and evolutionary studies. To make C. sp. 34 genetically tractable, we have been developing genetic and molecular techniques and tools. Stable transgenic lines of C. sp.34 could be obtained by microinjecting marker plasmids commonly used in C. elegans, although the efficiency was lower than that in C. elegans. Both soaking and feeding RNAi was as effective as in C. elegans. A panel of antibodies against C. elegans proteins successfully recognized expected structures in C. sp. 34 by immunofluorescence. Thus, many of the rich genetic and molecular resources for C. elegans can be directly used for C. sp. 34 studies. We well present some of the comparative analyses of gene functions regarding the body size, germ cell formation and sex determination.
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[
MicroPubl Biol,
2022]
The gonochoristic nematode Caenorhabditis inopinata is the phylogenetically closest species to the well-studied nematode Caenorhabditis elegans (Kanzaki et al. , 2018). While C. inopinata has been expected to be a useful comparative model for C. elegans , efficient transgenesis methods have not been available. Here, we established a method to integrate transgenes into the C. inopinata genome by microparticle bombardment with hygromycin B selection. C. elegans- derived genetic elements tested in this study, including universal and germline-specific promoters, ORFs, and 3'UTRs, were all functional in C. inopinata. Using this method, transgenic C. inopinata strains that express fluorescent subcellular markers were established.
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[
International Worm Meeting,
2013]
Since we reported on Caenorhabditis biodiversity in 2011, 13 new species have been discovered. The number of species in culture is now 36, and 50 species are known. Most new species were isolated from rotting plant material, but two were found in fresh figs (N. Kanzaki pers. comm.) and one in the hind gut of a millipede (W. Sudhaus pers. comm.). Three of the new species were isolated from temperate regions, the others from tropical regions. Preliminary phylogenetic analyses with molecular data for 36 species confirm the existence of two well-supported large sister clades, the Elegans super-group with now 21 species and the Drosophilae super-group with 11 species. C. plicata, C. sp. 1 and C. sonorae as well as a C. sp. 21 branch off basally. Still, no sister species of C. elegans has been found. Hybridization is now observed in crosses of 5 species pairs (C. angaria - C. sp. 12, C. briggsae - C. sp. 9, C. remanei - C. sp. 23, C. sp. 5 - C. sp. 26, C. sp. 8 - C. sp. 24), providing opportunity for studying the evolution of hybrid incompatibility in Caenorhabditis. In at least one case, crosses between individuals from some but not all populations show reproductive isolation, suggesting incomplete speciation. Using light and scanning electron microscopy, we are evaluating morphological characters of all cultured species in detail. Across Caenorhabditis, the morphological diversity is large, especially in features of the male tail and the stoma. However, no or only subtle differences are found between many species of the Elegans super-group. Mapping phenotypic characters onto the phylogeny shows extensive homoplasy (convergent evolution or secondary loss) across all character complexes. Morphological, biogeographical, ecological, sequence, and taxonomic data on all Caenorhabditis species is now available through an open-access online database RhabditinaDB
(http://wormtails.bio.nyu.edu/Databases). Strains of most species are available through the CGC.
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[
International Worm Meeting,
2015]
Since the 19th International C. elegans Meeting in 2013, discovery of new Caenorhabditis species has continued at a rapid rate. We now know of 63 species, 51 of which are in culture. All new species were found in tropical locations.A molecular phylogeny for 41 Caenorhabditis species was calculated with sequence data of 22 genes that were analyzed with three different algorithms. Previously reconstructed relationships within the genus are confirmed. Caenorhabditis contains three large monophyletic groups: The Drosophilae super group and the Japonica group and Elegans groups within the Elegans super group. Four species are not part of these clades and branch off early. Relationships within the Elegans super group are generally well resolved but the position of C. kamaaina remains uncertain. Two species isolated from fresh figs and likely associated with fig-wasps (Kanzaki pers. comm.) branch off as the sister group of C. elegans. The relationships within the Drosophilae super group are less well supported with conflicting placements of several subclades. We are currently incorporating the remaining species into the phylogeny.Light and scanning electron microscopic evaluation of morphology show that the diversity in phenotypic characters is large across the genus as a whole. However, most of this diversity is found in the Drosophilae super group and the basally branching species. Phenotypic diversity within the Elegans group is small in comparison. This is in contrast to the rate of molecular diversity, which is more uniform across all Caenorhabditis species. The analysis of phenotypic characters confirms that homoplasy is extensive and affects almost all characters studied.So far, genomes of 16 species have been sequenced. An initiative to sequence the genomes of all remaining Caenorhabditis species in culture was launched by Mark Blaxter and his lab in 2014
(http://caenorhabditis.bio.ed.ac.uk/).We continue to deposit morphological, biogeographical, ecological, sequence, and taxonomic data on all Caenorhabditis species in the open-access online database RhabditinaDB
(http://wormtails.bio.nyu.edu/Databases). Information about Caenorhabditis isolates is also found in a WIKI on WormBase
(http://evolution.wormbase.org/index.php/Main_Page). -
[
microPublication Biology,
2019]
Nematodes, such as the model organism Caenorhabditis elegans, communicate environmental and developmental information with conspecifics through a class of small-molecule pheromones termed ascarosides (Butcher, 2017; Chute and Srinivasan, 2014; Ludewig and Schroeder, 2013). Nematodes share ascaroside signaling pathways (Choe et al., 2012), but are also capable of eavesdropping on chemical signals of predatory species (Liu et al., 2018). Ascarosides signal vast arrays of information, either individually or as blends, based on concentration, sex, physiological state, and other ascarosides sensed (McGrath and Ruvinsky, 2019; Pungaliya et al., 2009; Srinivasan et al., 2008; Srinivasan et al., 2012). For instance, octopamine-succinylated ascaroside #9 (osas#9) is able to signal starvation conditions in the absence of other ascarosides (Artyukhin et al., 2013).C. elegans (Cel) is an androdioecious species, with the majority of the natural population comprised of self-fertilizing hermaphrodites, and a small proportion (<0.2%) being male (Hodgkin et al., 1979). There are two other similarly androdioecious species in the genus, C. briggsae (Cbr) and C. tropicalis (Ctr). All three species evolved their hermaphroditism separately and uniquely (Ellis and Lin, 2014). Of the male-attracting ascarosides secreted by C. elegans (ascr#2, ascr#3, ascr#4, and ascr#8), ascr#8 is the most potent (Pungaliya et al., 2009). Since ascr#8 is a male attractant in this hermaphroditic species, we asked if other hermaphroditic species retained the ability to attract males using this cue. Males from the gonochoristic (male-female) sister species to C. briggsae and C. tropicalis C. nigoni (Cni) and C. wallacei (Cwa), respectively were also assayed for their ability to respond to ascr#8. The closest relative of C. elegans, the gonochoristic C. inopinata (Cin, formerly C. sp. 34), which has been recently characterized (Kanzaki et al., 2018), was also tested, along with the JaponicaGroup gonochoristic species C. japonica(Cja) and C. afra(Caf).Dwell times were analyzed as previously described using a Spot Retention Assay (Narayan et al., 2016). Dwell times were transformed using a Base 2 Exponentiation (2n, wherein n is equal to the raw dwell time value) to generate only non-zero data in order to calculate fold-changes. The Logbase2 of the fold-changes was then calculated to normalize the data. All data sets were first checked for normality using a DAgostino