The contribution of an X chromosome QTL to non-Mendelian inheritance and unequal chromosomal segregation in Auanema freiburgense.

Auanema freiburgense is a nematode with males, females, and selfing hermaphrodites. When XO males mate with XX females, they typically produce a low proportion of XO offspring because they eliminate nullo-X spermatids. This process ensures that most sperm carry an X chromosome, increasing the likelihood of X chromosome transmission compared to random segregation. This occurs because of an unequal distribution of essential cellular organelles during sperm formation, likely dependent on the X chromosome. Some sperm components are selectively segregated into the X chromosome's daughter cell, while others are discarded with the nullo-X daughter cell. Intriguingly, the interbreeding of 2 A. freiburgense strains results in hybrid males capable of producing viable nullo-X sperm. Consequently, when these hybrid males mate with females, they yield a high percentage of male offspring. To uncover the genetic basis of nullo-spermatid elimination and X chromosome drive, we generated a genome assembly for A. freiburgense and genotyped the intercrossed lines. This analysis identified a quantitative trait locus spanning several X chromosome genes linked to the non-Mendelian inheritance patterns observed in A. freiburgense. This finding provides valuable clues to the underlying factors involved in asymmetric organelle partitioning during male meiotic division and thus non-Mendelian transmission of the X chromosome and sex ratios.

SKN-1/NRF2 upregulation by vitamin A is conserved from nematodes to mammals and is critical for lifespan extension in Caenorhabditis elegans.

Vitamin A (VA) is a micronutrient essential for the physiology of many organisms, but its role in longevity and age-related diseases remains unclear. In this work, we used Caenorhabditis elegans to study the impact of various bioactive compounds on lifespan. We demonstrate that VA extends lifespan and reduces lipofuscin and fat accumulation while increasing resistance to heat and oxidative stress. This resistance can be attributed to high levels of detoxifying enzymes called glutathione S-transferases, induced by the transcription factor skinhead-1 (SKN-1). Notably, VA upregulated the transcript levels of skn-1 or its mammalian ortholog NRF2 in both C. elegans, human cells, and liver tissues of mice. Moreover, the loss-of-function genetic models demonstrated a critical involvement of the SKN-1 pathway in longevity extension by VA. Our study thus provides novel insights into the molecular mechanism of anti-aging and anti-oxidative effects of VA, suggesting that this micronutrient could be used for the prevention and/or treatment of age-related disorders.

Lagging X chromatids specify the orientation of asymmetric organelle partitioning in XX spermatocytes of Auanema rhodensis.

The unequal partitioning of molecules and organelles during cell division results in daughter cells with different fates. An extreme example is female meiosis, in which consecutive asymmetric cell divisions give rise to 1 large oocyte and 2 small polar bodies with DNA and minimal cytoplasm. Here, we test the hypothesis that during an asymmetric cell division during spermatogenesis of the nematode Auanema rhodensis, the late segregating X chromatids orient the asymmetric partitioning of cytoplasmic components. In previous studies, the secondary spermatocytes of wild-type XO males were found to divide asymmetrically to generate functional spermatids that inherit components necessary for sperm viability and DNA-containing residual bodies that inherit components to be discarded. Here we extend that analysis to 2 novel contexts. First, the isolation and analysis of a strain of mutant XX pseudomales revealed that such animals have highly variable patterns of X-chromatid segregation. The pattern of late segregating X chromatids nevertheless predicted the orientation of organelle partitioning. Second, while wild-type XX hermaphrodites were known to produce both 1X and 2X sperm, here, we show that spermatocytes within specific spermatogonial clusters exhibit 2 different patterns of X-chromatid segregation that correlate with distinct patterns of organelle partitioning. Together this analysis suggests that A. rhodensis has coopted lagging X chromosomes during anaphase II as a mechanism for determining the orientation of organelle partitioning.

Sexual morph specialisation in a trioecious nematode balances opposing selective forces.

The coexistence of different mating strategies, whereby a species can reproduce both by selfing and outcrossing, is an evolutionary enigma. Theory predicts two predominant stable mating states: outcrossing with strong inbreeding depression or selfing with weak inbreeding depression. As these two mating strategies are subject to opposing selective forces, mixed breeding systems are thought to be a rare transitory state yet can persist even after multiple speciation events. We hypothesise that if each mating strategy plays a distinctive role during some part of the species life history, opposing selective pressures could be balanced, permitting the stable co-existence of selfing and outcrossing sexual morphs. In this scenario, we would expect each morph to be specialised in their respective roles. Here we show, using behavioural, physiological and gene expression studies, that the selfing (hermaphrodite) and outcrossing (female) sexual morphs of the trioecious nematode Auanema freiburgensis have distinct adaptations optimised for their different roles during the life cycle. A. freiburgensis hermaphrodites are known to be produced under stressful conditions and are specialised for dispersal to new habitat patches. Here we show that they exhibit metabolic and intestinal changes enabling them to meet the cost of dispersal and reproduction. In contrast, A. freiburgensis females are produced in favourable conditions and facilitate rapid population growth. We found that females compensate for the lack of reproductive assurance by reallocating resources from intestinal development to mate-finding behaviour. The specialisation of each mating system for its role in the life cycle could balance opposing selective forces allowing the stable maintenance of both mating systems in A. freiburgensis.

De Novo Genome Assembly of Auanema Melissensis, a Trioecious Free-Living Nematode.

Nematodes of the genus Auanema are interesting models for studying sex determination mechanisms because their populations consist of three sexual morphs (males, females, and hermaphrodites) and produce skewed sex ratios. Here, we introduce a new undescribed species of this genus, Auanema melissensis n. sp., together with its draft nuclear genome. This species is also trioecious and does not cross with the other described species A. rhodensis or A. freiburgensis. Similar to A. freiburgensis, A. melissensis' maternal environment influences the hermaphrodite versus female sex determination of the offspring. The genome of A. melissensis is ~60 Mb, containing 11,040 protein-coding genes and 8.07% of repeat sequences. Using the estimated ancestral chromosomal gene content (Nigon elements), it was possible to identify putative X chromosome scaffolds.

Parental energy-sensing pathways control intergenerational offspring sex determination in the nematode Auanema freiburgensis.

BACKGROUND: Environmental stimuli experienced by the parental generation influence the phenotype of subsequent generations (Demoinet et al., Proc Natl Acad Sci U S A 114:E2689-E2698, 2017; Burton et al., Nat Cell Biol 19:252-257, 2017; Agrawal et al., Nature 401:60-63, 1999). The effects of these stimuli on the parental generation may be passed through the germline, but the mechanisms at the basis of this non-Mendelian type of inheritance, their level of conservation, how they lead to adaptive vs non-adaptive, and intergenerational vs transgenerational inheritance are poorly understood. Here we show that modulation of nutrient-sensing pathways in the parental generation of the nematode Auanema freiburgensis regulates phenotypic plasticity of its offspring. RESULTS: In response to con-specific pheromones indicative of stress, AMP-activated protein kinase (AMPK), mechanistic target of rapamycin complex 1 (mTORC1), and insulin signaling regulate stress resistance and sex determination across one generation, and these effects can be mimicked by pathway modulators. The effectors of these pathways are closely associated with the chromatin, and their regulation affects the chromatin acetylation status in the germline. CONCLUSION: These results suggest that highly conserved metabolic sensors regulate phenotypic plasticity through regulation of subcellular localization of their effectors, leading to changes in chromatin acetylation and epigenetic status of the germline.

Toward genetic modification of plant-parasitic nematodes: delivery of macromolecules to adults and expression of exogenous mRNA in second stage juveniles.

Plant-parasitic nematodes are a continuing threat to food security, causing an estimated 100 billion USD in crop losses each year. The most problematic are the obligate sedentary endoparasites (primarily root knot nematodes and cyst nematodes). Progress in understanding their biology is held back by a lack of tools for functional genetics: forward genetics is largely restricted to studies of natural variation in populations and reverse genetics is entirely reliant on RNA interference. There is an expectation that the development of functional genetic tools would accelerate the progress of research on plant-parasitic nematodes, and hence the development of novel control solutions. Here, we develop some of the foundational biology required to deliver a functional genetic tool kit in plant-parasitic nematodes. We characterize the gonads of male Heterodera schachtii and Meloidogyne hapla in the context of spermatogenesis. We test and optimize various methods for the delivery, expression, and/or detection of exogenous nucleic acids in plant-parasitic nematodes. We demonstrate that delivery of macromolecules to cyst and root knot nematode male germlines is difficult, but possible. Similarly, we demonstrate the delivery of oligonucleotides to root knot nematode gametes. Finally, we develop a transient expression system in plant-parasitic nematodes by demonstrating the delivery and expression of exogenous mRNA encoding various reporter genes throughout the body of H. schachtii juveniles using lipofectamine-based transfection. We anticipate these developments to be independently useful, will expedite the development of genetic modification tools for plant-parasitic nematodes, and ultimately catalyze research on a group of nematodes that threaten global food security.

Newly Identified Nematodes from Mono Lake Exhibit Extreme Arsenic Resistance.

Extremophiles have much to reveal about the biology of resilience, yet their study is limited by sampling and culturing difficulties [1-3]. The broad success and small size of nematodes make them advantageous for tackling these problems [4-6]. We investigated the arsenic-rich, alkaline, and hypersaline Mono Lake (CA, US) [7-9] for extremophile nematodes. Though Mono Lake has previously been described to contain only two animal species (brine shrimp and alkali flies) in its water and sediments [10], we report the discovery of eight nematode species from the lake, including microbe grazers, parasites, and predators. Thus, nematodes are the dominant animals of Mono Lake in species richness. Phylogenetic analysis suggests that the nematodes originated from multiple colonization events, which is striking, given the young history of extreme conditions at Mono Lake [7, 11]. One species, Auanema sp., is new, culturable, and survives 500 times the human lethal dose of arsenic. Comparisons to two non-extremophile sister species [12] reveal that arsenic resistance is a common feature of the genus and a preadaptive trait that likely allowed Auanema to inhabit Mono Lake. This preadaptation may be partly explained by a variant in the gene dbt-1 shared with some Caenorhabditis elegans natural populations and known to confer arsenic resistance [13]. Our findings expand Mono Lake's ecosystem from two known animal species to ten, and they provide a new system for studying arsenic resistance. The dominance of nematodes in Mono Lake and other extreme environments and our findings of preadaptation to arsenic raise the intriguing possibility that nematodes are widely pre-adapted to be extremophiles.

Chromosome-Wide Evolution and Sex Determination in the Three-Sexed Nematode Auanema rhodensis.

Trioecy, a mating system in which males, females and hermaphrodites co-exist, is a useful system to investigate the origin and maintenance of alternative mating strategies. In the trioecious nematode Auanema rhodensis, males have one X chromosome (XO), whereas females and hermaphrodites have two (XX). The female vs. hermaphrodite sex determination mechanisms have remained elusive. In this study, RNA-seq analyses show a 20% difference between the L2 hermaphrodite and female gene expression profiles. RNAi experiments targeting the DM (doublesex/mab-3) domain transcription factor dmd-10/11 suggest that the hermaphrodite sexual fate requires the upregulation of this gene. The genetic linkage map (GLM) shows that there is chromosome-wide heterozygosity for the X chromosome in F2 hermaphrodite-derived lines originated from crosses between two parental inbred strains. These results confirm the lack of recombination of the X chromosome in hermaphrodites, as previously reported. We also describe conserved chromosome elements (Nigon elements), which have been mostly maintained throughout the evolution of Rhabditina nematodes. The seven-chromosome karyotype of A. rhodensis, instead of the typical six found in other rhabditine species, derives from fusion/rearrangements events involving three Nigon elements. The A. rhodensis X chromosome is the smallest and most polymorphic with the least proportion of conserved genes. This may reflect its atypical mode of father-to-son transmission and its lack of recombination in hermaphrodites and males. In conclusion, this study provides a framework for studying the evolution of chromosomes in rhabditine nematodes, as well as possible mechanisms for the sex determination in a three-sexed species.

Liposome-based transfection enhances RNAi and CRISPR-mediated mutagenesis in non-model nematode systems.

Nematodes belong to one of the most diverse animal phyla. However, functional genomic studies in nematodes, other than in a few species, have often been limited in their reliability and success. Here we report that by combining liposome-based technology with microinjection, we were able to establish a wide range of genomic techniques in the newly described nematode genus Auanema. The method also allowed heritable changes in dauer larvae of Auanema, despite the immaturity of the gonad at the time of the microinjection. As proof of concept for potential functional studies in other nematode species, we also induced RNAi in the free-living nematode Pristionchus pacificus and targeted the human parasite Strongyloides stercoralis.

Sex- and Gamete-Specific Patterns of X Chromosome Segregation in a Trioecious Nematode.

Three key steps in meiosis allow diploid organisms to produce haploid gametes: (1) homologous chromosomes (homologs) pair and undergo crossovers; (2) homologs segregate to opposite poles; and (3) sister chromatids segregate to opposite poles. The XX/XO sex determination system found in many nematodes [1] facilitates the study of meiosis because variation is easily recognized [2-4]. Here we show that meiotic segregation of X chromosomes in the trioecious nematode Auanema rhodensis [5] varies according to sex (hermaphrodite, female, or male) and type of gametogenesis (oogenesis or spermatogenesis). In this species, XO males exclusively produce X-bearing sperm [6, 7]. The unpaired X precociously separates into sister chromatids, which co-segregate with the autosome set to generate a functional haplo-X sperm. The other set of autosomes is discarded into a residual body. Here we explore the X chromosome behavior in female and hermaphrodite meioses. Whereas X chromosomes segregate following the canonical pattern during XX female oogenesis to yield haplo-X oocytes, during XX hermaphrodite oogenesis they segregate to the first polar body to yield nullo-X oocytes. Thus, crosses between XX hermaphrodites and males yield exclusively male progeny. During hermaphrodite spermatogenesis, the sister chromatids of the X chromosomes separate during meiosis I, and homologous X chromatids segregate to the functional sperm to create diplo-X sperm. Given these intra-species, intra-individual, and intra-gametogenesis variations in the meiotic program, A. rhodensis is an ideal model for studying the plasticity of meiosis and how it can be modulated.

Description of two three-gendered nematode species in the new genus Auanema (Rhabditina) that are models for reproductive mode evolution.

The co-existence of males, females and hermaphrodites, a rare mating system known as trioecy, has been considered as an evolutionarily transient state. In nematodes, androdioecy (males/hermaphrodites) as found in Caenorhabditis elegans, is thought to have evolved from dioecy (males/females) through a trioecious intermediate. Thus, trioecious species are good models to understand the steps and requirements for the evolution of new mating systems. Here we describe two new species of nematodes with trioecy, Auanema rhodensis and A. freiburgensis. Along with molecular barcodes, we provide a detailed analysis of the morphology of these species, and document it with drawings and light and SEM micrographs. Based on morphological data, these free-living nematodes were assigned to a new genus, Auanema, together with three other species described previously. Auanema species display convergent evolution in some features with parasitic nematodes with complex life cycles, such as the production of few males after outcrossing and the obligatory development of dauers into self-propagating adults.

Cytoskeletal variations in an asymmetric cell division support diversity in nematode sperm size and sex ratios.

Asymmetric partitioning is an essential component of many developmental processes. As spermatogenesis concludes, sperm are streamlined by discarding unnecessary cellular components into cellular wastebags called residual bodies (RBs). During nematode spermatogenesis, this asymmetric partitioning event occurs shortly after anaphase II, and both microtubules and actin partition into a central RB. Here, we use fluorescence and transmission electron microscopy to elucidate and compare the intermediate steps of RB formation in Caenorhabditis elegans, Rhabditis sp. SB347 (recently named Auanema rhodensis) and related nematodes. In all cases, intact microtubules reorganize and move from centrosomal to non-centrosomal sites at the RB-sperm boundary whereas actin reorganizes through cortical ring expansion and clearance from the poles. However, in species with tiny spermatocytes, these cytoskeletal changes are restricted to one pole. Consequently, partitioning yields one functional sperm with the X-bearing chromosome complement and an RB with the other chromosome set. Unipolar partitioning may not require an unpaired X, as it also occurs in XX spermatocytes. Instead, constraints related to spermatocyte downsizing may have contributed to the evolution of a sperm cell equivalent to female polar bodies.

Sex-specific lifespan and its evolution in nematodes.

Differences between sexes of the same species in lifespan and aging rate are widespread. While the proximal and evolutionary causes of aging are well researched, the factors that contribute to sex differences in these traits have been less studied. The striking diversity of nematodes provides ample opportunity to study variation in sex-specific lifespan patterns associated with shifts in life history and mating strategy. Although the plasticity of these sex differences will make it challenging to generalize from invertebrate to vertebrate systems, studies in nematodes have enabled empirical evaluation of predictions regarding the evolution of lifespan. These studies have highlighted how natural and sexual selection can generate divergent patterns of lifespan if the sexes are subject to different rates or sources of mortality, or if trade-offs between complex traits and longevity are resolved differently in each sex. Here, we integrate evidence derived mainly from nematodes that addresses the molecular and evolutionary basis of sex-specific aging and lifespan. Ultimately, we hope to generate a clearer picture of current knowledge in this area, and also highlight the limitations of our understanding.

Phenotypic plasticity and developmental innovations in nematodes.

Developmental plasticity has been implicated as a facilitator for phenotypic diversification, but the molecular mechanisms controlling it are largely unknown. We review recent comparative analyses in non-Caenorhabditis nematodes that display polyphenisms in larval development, mouth morphology and reproductive mode. Some of the challenges ahead will be to connect how these phenotypic traits are linked to each other at the molecular level, and at the ecological level. This will require sampling of several nematode species, the characterization of their ecology and the employment of both classical genetics and recently developed technological advances, such as genome editing.

Mating dynamics in a nematode with three sexes and its evolutionary implications.

Nematodes have diverse reproductive strategies, which make them ideal subjects for comparative studies to address how mating systems evolve. Here we present the sex ratios and mating dynamics of the free-living nematode Rhabditis sp. SB347, in which males, females and hermaphrodites co-exist. The three sexes are produced by both selfing and outcrossing, and females tend to appear early in a mother's progeny. Males prefer mating with females over hermaphrodites, which our results suggest is related to the female-specific production of the sex pheromones ascr#1 and ascr#9. We discuss the parallels between this system and that of parasitic nematodes that exhibit alternation between uniparental and biparental reproduction.

The genome of Tetranychus urticae reveals herbivorous pest adaptations.

The spider mite Tetranychus urticae is a cosmopolitan agricultural pest with an extensive host plant range and an extreme record of pesticide resistance. Here we present the completely sequenced and annotated spider mite genome, representing the first complete chelicerate genome. At 90 megabases T. urticae has the smallest sequenced arthropod genome. Compared with other arthropods, the spider mite genome shows unique changes in the hormonal environment and organization of the Hox complex, and also reveals evolutionary innovation of silk production. We find strong signatures of polyphagy and detoxification in gene families associated with feeding on different hosts and in new gene families acquired by lateral gene transfer. Deep transcriptome analysis of mites feeding on different plants shows how this pest responds to a changing host environment. The T. urticae genome thus offers new insights into arthropod evolution and plant-herbivore interactions, and provides unique opportunities for developing novel plant protection strategies.