Molecular cloning, identification, transcriptional analysis, and silencing of enolase on the life cycle of Haemaphysalis longicornis (Acari, Ixodidae) tick.

Ticks, blood-sucking ectoparasites, spread diseases to humans and animals. Haemaphysalis longicornis is a significant vector for tick-borne diseases in medical and veterinary contexts. Identifying protective antigens in H. longicornis for an anti-tick vaccine is a key tick control strategy. Enolase, a multifunctional protein, significantly converts D-2-phosphoglycerate and phosphoenolpyruvate in glycolysis and gluconeogenesis in cell cytoplasm. This study cloned a complete open reading frame (ORF) of enolase from the H. longicornis tick and characterized its transcriptional and silencing effect. We amplified the full-length cDNA of the enolase gene using rapid amplification of cDNA ends. The complete cDNA, with an ORF of 1,297 nucleotides, encoded a 432-amino acid polypeptide. Enolase of the Jeju strain H. longicornis exhibited the highest sequence similarity with H. flava (98%), followed by Dermacentor silvarum (82%). The enolase motifs identified included N-terminal and C-terminal regions, magnesium binding sites, and several phosphorylation sites. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that enolase mRNA transcripts were expressed across all developmental stages of ticks and organs such as salivary gland and midgut. RT-PCR showed higher transcript levels in syn-ganglia, suggesting that synganglion nerves influence enolase,s role in tick salivary glands. We injected enolase double-stranded RNA into adult unfed female ticks, after which they were subsequently fed with normal unfed males until they spontaneously dropped off. RNA interference significantly (P<0.05) reduced feeding and reproduction, along with abnormalities in eggs (no embryos) and hatching. These findings suggest enolase is a promising target for future tick control strategies.

The DEAD-box RNA helicase PfDOZI imposes opposing actions on RNA metabolism in Plasmodium falciparum.

In malaria parasites, the regulation of mRNA translation, storage and degradation during development and life-stage transitions remains largely unknown. Here, we functionally characterized the DEAD-box RNA helicase PfDOZI in P. falciparum. Disruption of pfdozi enhanced asexual proliferation but reduced sexual commitment and impaired gametocyte development. By quantitative transcriptomics, we show that PfDOZI is involved in the regulation of invasion-related genes and sexual stage-specific genes during different developmental stages. PfDOZI predominantly participates in processing body-like mRNPs in schizonts but germ cell granule-like mRNPs in gametocytes to impose opposing actions of degradation and protection on different mRNA targets. We further show the formation of stress granule-like mRNPs during nutritional deprivation, highlighting an essential role of PfDOZI-associated mRNPs in stress response. We demonstrate that PfDOZI participates in distinct mRNPs to maintain mRNA homeostasis in response to life-stage transition and environmental changes by differentially executing post-transcriptional regulation on the target mRNAs.

Transcriptional control of the Cryptosporidium life cycle.

The parasite Cryptosporidium is a leading agent of diarrhoeal disease in young children, and a cause and consequence of chronic malnutrition1,2. There are no vaccines and only limited treatment options3. The parasite infects enterocytes, in which it engages in asexual and sexual replication4, both of which are essential to continued infection and transmission. However, their molecular mechanisms remain largely unclear5. Here we use single-cell RNA sequencing to reveal the gene expression programme of the entire Cryptosporidium parvum life cycle in culture and in infected animals. Diverging from the prevailing model6, we find support for only three intracellular stages: asexual type-I meronts, male gamonts and female gametes. We reveal a highly organized program for the assembly of components at each stage. Dissecting the underlying regulatory network, we identify the transcription factor Myb-M as the earliest determinant of male fate, in an organism that lacks genetic sex determination. Conditional expression of this factor overrides the developmental program and induces widespread maleness, while conditional deletion ablates male development. Both have a profound impact on the infection. A large set of stage-specific genes now provides the opportunity to understand, engineer and disrupt parasite sex and life cycle progression to advance the development of vaccines and treatments.

Transcriptomic analysis of N-terminal mutated Trypanosoma cruzi UBP1 knockdown underlines the importance of this RNA-binding protein in parasite development.

BACKGROUND: During its life cycle, the human pathogen Trypanosoma cruzi must quickly adapt to different environments, in which the variation in the gene expression of the regulatory U-rich RNA-binding protein 1 (TcUBP1) plays a crucial role. We have previously demonstrated that the overexpression of TcUBP1 in insect-dwelling epimastigotes orchestrates an RNA regulon to promote differentiation to infective forms. METHODS: In an attempt to generate TcUBP1 knockout parasites by using CRISPR-Cas9 technology, in the present study, we obtained a variant transcript that encodes a protein with 95% overall identity and a modified N-terminal sequence. The expression of this mutant protein, named TcUBP1mut, was notably reduced compared to that of the endogenous form found in normal cells. TcUBP1mut-knockdown epimastigotes exhibited normal growth and differentiation into infective metacyclic trypomastigotes and were capable of infecting mammalian cells. RESULTS: We analyzed the RNA-Seq expression profiles of these parasites and identified 276 up- and 426 downregulated genes with respect to the wildtype control sample. RNA-Seq comparison across distinct developmental stages revealed that the transcriptomic profile of these TcUBP1mut-knockdown epimastigotes significantly differs not only from that of epimastigotes in the stationary phase but also from the gene expression landscape characteristic of infective forms. This is both contrary to and consistent with the results of our recent study involving TcUBP1-overexpressing cells. CONCLUSION: Together, our findings demonstrate that the genes exhibiting opposite changes under overexpression and knockdown conditions unveil key mRNA targets regulated by TcUBP1. These mostly encompass transcripts that encode for trypomastigote-specific surface glycoproteins and ribosomal proteins, supporting a role for TcUBP1 in determining the molecular characteristics of the infective stage.

From a bimodal to a multi-stage view on trypanosomes' differential RNA editing.

Significant variations in the abundance of mitochondrial RNA processing proteins and their target RNAs across trypanosome life stages present an opportunity to explore the regulatory mechanisms that drive these changes. Utilizing omics approaches can uncover unconventional targets, aiding our understanding of the parasites' adaptation and enabling targeted interventions for differentiation.

Transcriptome variation in banded newt (Ommatotriton vittatus) during its life cycle and habitat transition.

Israel represents the southern limit of the distribution of the banded newt (Ommatotriton vittatus). The life cycle of O. vittatus includes several distinct phases: eggs, aquatic larvae, a terrestrial phase and an aquatic reproductive phase. We investigated differences in gene expression during the life cycle and transition of banded newts between terrestrial and aquatic habitats using mRNA-seq. We identified ∼10 k genes that were differentially expressed (DE) in one of the pairwise comparisons between 3 groups: 1 - terrestrial newts (males and females), 2 - aquatic newts (males and females), 3 - aquatic larvae before metamorphosis. The groups were clearly defined by Principal Components Analysis (PCA). The greatest difference was between aquatic newts (males and females) and aquatic larvae: ∼7.4 k DE genes. Of special interest were the ∼2.4 k genes DE between the aquatic and terrestrial phenotypes. These included prominent candidates with known roles in kidney function (uromodulin homologs were strongly associated with aquatic lifestyle), tissue structure (keratins), and the thyroid hormone signaling modulator DUOXA1. Additional developmental and metabolic pathways overrepresented among the identified DE genes included "epidermis development", "nervous system development", "nucleotide-sugar biosynthesis". Overall, both metamorphosis and environmental transition of banded newts involve extensive transcriptomic remodeling involving developmental, metabolic, and cellular pathways. Understanding the roles of these pathways and individual genes is instrumental for studies of transition between habitats, especially those affected by climate change. Furthermore, the phenotypic flexibility of the newt and the underlying regulation of gene expression can shed light on the evolution of terrestrial vertebrates.

Comprehensive analysis of miRNA profiling in Schistosoma mekongi across life cycle stages.

Schistosoma mekongi, a significant schistosome parasite, has various life stages, including egg, cercaria, female, and male, that play crucial roles in the complex life cycle. This study aimed to explore the microRNA (miRNA) profiles across these developmental stages to understand their potential functions and evolutionary significance, which have not been studied. Pre-processed sequencing reads of small RNA (sRNA) were obtained, and annotations were performed against the S. japonicum reference miRNA database. Results indicated marked variations in miRNA profiles across different life stages, with notable similarities observed between female and male S. mekongi. Principal Coordinate Analysis (PCoA) and unsupervised clustering revealed distinct miRNA signatures for each stage. Gene ontology (GO) analysis unveiled the potential roles of these miRNAs in various biological processes. The differential expression of specific miRNAs was prominent across stages, suggesting their involvement in crucial developmental processes. Furthermore, orthologous miRNA analysis against various worm species revealed distinct presence-absence patterns, providing insights into the evolutionary relationships of these miRNAs. In conclusion, this comprehensive investigation into the miRNA profiles of S. mekongi offers valuable insights into the functional and evolutionary aspects of miRNAs in schistosome biology.

In vitro production of cat-restricted Toxoplasma pre-sexual stages.

Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described1,2. Here we found that the transcription factors AP2XII-1 and AP2XI-2 operate during the tachyzoite stage, a hallmark of acute toxoplasmosis, to silence genes necessary for merozoites, a developmental stage critical for subsequent sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a marked change in the transcriptional program, promoting a full transition from tachyzoites to merozoites. These in vitro-cultured pre-gametes have unique protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit MORC and HDAC3 (ref. 1), thereby limiting chromatin accessibility and transcription. Consequently, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. Successful production of merozoites in vitro paves the way for future studies on Toxoplasma sexual development without the need for cat infections and holds promise for the development of therapies to prevent parasite transmission.

Differential expression of meiosis and homologous recombination-related genes in the life cycle of Trypanosoma cruzi.

Trypanosoma cruzi has a complex life cycle consisting of four morphological and distinct biological stages. Although some authors suggest that T. cruzi primarily follows clonal reproduction, recent genomic and transcriptomic studies indicate an unorthodox capacity for recombination. We aimed to estimate the differential gene expression of 10 meiosis/homologous recombination-related genes during the T. cruzi life cycle, including epimastigotes, under two different types of stress (oxidative stress and pH changes). We performed RT-qPCR tests using novel-designed primers to estimate the differential gene expression (∆Ct and ∆∆Ct) of nine genes (SPO11, HAP2, RAD50, MRN complex, BRCA2, DMC1, MND1, and RPA1) and RAD51, which was previously reported. Our results show basal expression of all genes during the life cycle, indicating their hypothetical role in several cellular processes but with specific signatures of differential gene expression during the life cycle (HAP2, RPA, RAD50, BRCA2, MND1, and DMC1) and oxidative stress (RPA, MRE11, NBS1, BRCA2, MND1, and RAD51). Additionally, we found that the MRN complex has an independent level of expression in T. cruzi, with profiles of MRE11 and NBS1 upregulated in some stages. Recent studies on other trypanosomatids have highlighted the influence of HAP2 and RPA in recombination and hybridization. If T. cruzi uses the same repertoire of genes, our findings could suggest that metacyclogenesis may be the putative step that the parasite uses to undergo recombination. Likewise, our study reveals the differential profiles of genes expressed in response to oxidative and pH stress. Further studies are necessary to confirm our findings and understand the recombination mechanism in T. cruzi.

Low Spontaneous Mutation Rate in Complex Multicellular Eukaryotes with a Haploid-Diploid Life Cycle.

The spontaneous mutation rate µ is a crucial parameter to understand evolution and biodiversity. Mutation rates are highly variable across species, suggesting that µ is susceptible to selection and drift and that species life cycle and life history may impact its evolution. In particular, asexual reproduction and haploid selection are expected to affect the mutation rate, but very little empirical data are available to test this expectation. Here, we sequence 30 genomes of a parent-offspring pedigree in the model brown alga Ectocarpus sp.7, and 137 genomes of an interspecific cross of the closely related brown alga Scytosiphon to have access to the spontaneous mutation rate of representative organisms of a complex multicellular eukaryotic lineage outside animals and plants, and to evaluate the potential impact of life cycle on the mutation rate. Brown algae alternate between a haploid and a diploid stage, both multicellular and free living, and utilize both sexual and asexual reproduction. They are, therefore, excellent models to empirically test expectations of the effect of asexual reproduction and haploid selection on mutation rate evolution. We estimate that Ectocarpus has a base substitution rate of µbs = 4.07 × 10-10 per site per generation, whereas the Scytosiphon interspecific cross had µbs = 1.22 × 10-9. Overall, our estimations suggest that these brown algae, despite being multicellular complex eukaryotes, have unusually low mutation rates. In Ectocarpus, effective population size (Ne) could not entirely explain the low µbs. We propose that the haploid-diploid life cycle, combined with extensive asexual reproduction, may be additional key drivers of the mutation rate in these organisms.

Odd family reunion: DNA barcoding reveals unexpected relationship between three hydrozoan species.

Knowledge of life histories is crucial for understanding ecological and evolutionary processes, but for many hydrozoan species only incomplete life cycles have been described due to challenges in linking hydromedusae with their polyp stages. Using a combination of DNA barcoding, morphology, and ecological information, we describe for the first time the polyp stage of Halopsis ocellata Agassiz, 1865 and re-describe that of Mitrocomella polydiademata (Romanes, 1876). Campanulinid hydroids referable to Lafoeina tenuis Sars, 1874 and collected in the same biogeographic region as the type locality of this species are shown to be the polyp stage of these two mitrocomid hydromedusae. The nominal species L. tenuis thus is a species complex that includes the polyp stage of medusae belonging to at least two genera currently placed in a different family. Consistent morphological and ecological differences were found between the polyps linked to each of these two hydromedusae, but molecular results suggest that yet other species may have morphologically similar hydroids. Polyps morphologically identified to L. tenuis are therefore better referred to as Lafoeina tenuis-type until further associations are resolved, particularly when occurring outside of the area of distribution of H. ocellata and M. polydiademata. Molecular identification integrated with traditional taxonomy is confirmed as an effective approach to link inconspicuous stages of marine invertebrates with hitherto unknown life cycles, especially in often-overlooked taxa. Disentangling the relationships between L. tenuis, H. ocellata, and M. polydiademata lays the ground for future research aimed at resolving the taxonomy and systematics of the enigmatic families Mitrocomidae and Campanulinidae.

Transcriptomic analysis of the adaptation to prolonged starvation of the insect-dwelling Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi is a digenetic unicellular parasite that alternates between a blood-sucking insect and a mammalian, host causing Chagas disease or American trypanosomiasis. In the insect gut, the parasite differentiates from the non-replicative trypomastigote forms that arrive upon blood ingestion to the non-infective replicative epimastigote forms. Epimastigotes develop into infective non-replicative metacyclic trypomastigotes in the rectum and are delivered via the feces. In addition to these parasite stages, transitional forms have been reported. The insect-feeding behavior, characterized by few meals of large blood amounts followed by long periods of starvation, impacts the parasite population density and differentiation, increasing the transitional forms while diminishing both epimastigotes and metacyclic trypomastigotes. To understand the molecular changes caused by nutritional restrictions in the insect host, mid-exponentially growing axenic epimastigotes were cultured for more than 30 days without nutrient supplementation (prolonged starvation). We found that the parasite population in the stationary phase maintains a long period characterized by a total RNA content three times smaller than that of exponentially growing epimastigotes and a distinctive transcriptomic profile. Among the transcriptomic changes induced by nutrient restriction, we found differentially expressed genes related to managing protein quality or content, the reported switch from glucose to amino acid consumption, redox challenge, and surface proteins. The contractile vacuole and reservosomes appeared as cellular components enriched when ontology term overrepresentation analysis was carried out, highlighting the roles of these organelles in starving conditions possibly related to their functions in regulating cell volume and osmoregulation as well as metabolic homeostasis. Consistent with the quiescent status derived from nutrient restriction, genes related to DNA metabolism are regulated during the stationary phase. In addition, we observed differentially expressed genes related to the unique parasite mitochondria. Finally, our study identifies gene expression changes that characterize transitional parasite forms enriched by nutrient restriction. The analysis of the here-disclosed regulated genes and metabolic pathways aims to contribute to the understanding of the molecular changes that this unicellular parasite undergoes in the insect vector.

How larvae and life cycles evolve.

Marine larvae have factored heavily in pursuits to understand the origin and evolution of animal life cycles. Recent comparisons of gene expression and chromatin state in different species of sea urchin and annelid show how evolutionary changes in embryonic gene regulation can lead to markedly different larval forms.

Identification of three members of the multidomain adhesion CCp family in Babesia gibsoni.

Babesia gibsoni is an intraerythrocytic apicomplexan parasite transmitted by Haemaphysalis longicornis and causes canine babesiosis. Within the tick, the Babesia parasite undergoes sexual conjugation and the sporogony process of its life cycle. To control B. gibsoni infection, prompt and effective treatment of acute infections and curing chronic carriers are urgently needed. Gene disruption of Plasmodium CCps resulted in blocking the transition of sporozoites from the mosquito midgut to the salivary glands, showing that these proteins are potential targets for the development of a transmission-blocking vaccine. In this study, we described the identification and characterization of three members of the CCp family in B. gibsoni, named CCp1, CCp2, and CCp3. The B. gibsoni sexual stages were induced in vitro by exposing parasites to xanthurenic acid (XA), dithiothreitol (DTT), and tris (2-carboxyethyl) phosphine (TCEP) at serial concentrations. Among them, 100 µM XA-exposed and cultured at 27 °C without CO2B. gibsoni presented diverse morphologies, including parasites with long projections, gradually increased free merozoites, and aggregated and round forms, indicative of sexual stage induction. Then, the expression of CCp proteins of induced parasites was confirmed by real-time reverse transcription PCR, immunofluorescence, and western blot. The results showed that BgCCp genes were highly significantly increased at 24 h post-sexual stage induction (p < 0.01). The induced parasites were recognized by anti-CCp mouse antisera and anti-CCp 1, 2, and 3 antibodies weakly reacted with sexual stage proteins of expected molecular weights of 179.4, 169.8, and 140.0 KDa, respectively. Our observations on morphological changes and confirmation of sexual stage protein expression will advance elemental biological research and lay the foundation for the development of transmission-blocking vaccines against canine babesiosis.

Toxoplasma gondii AP2XII-2 Contributes to Transcriptional Repression for Sexual Commitment.

Toxoplasma gondii is a widespread protozoan parasite that has a significant impact on human and veterinary health. The parasite undergoes a complex life cycle involving multiple hosts and developmental stages. How Toxoplasma transitions between life cycle stages is poorly understood yet central to controlling transmission. Of particular neglect are the factors that contribute to its sexual development, which takes place exclusively in feline intestines. While epigenetic repressors have been shown to play an important role in silencing the spurious gene expression of sexually committed parasites, the specific factors that recruit this generalized machinery to the appropriate genes remain largely unexplored. Here, we establish that a member of the AP2 transcription factor family, AP2XII-2, is targeted to genomic loci associated with sexually committed parasites along with epigenetic regulators of transcriptional silencing, HDAC3 and MORC. Despite its widespread association with gene promoters, AP2XII-2 is required for the silencing of relatively few genes. Using the CUT&Tag (cleavage under targets and tagmentation) methodology, we identify two major genes associated with sexual development downstream of AP2XII-2 control, AP2X-10 and the amino acid hydroxylase AAH1. Our findings show that AP2XII-2 is a key contributor to the gene regulatory pathways modulating Toxoplasma sexual development. IMPORTANCE Toxoplasma gondii is a parasite that undergoes its sexual stage exclusively in feline intestines, making cats a major source of transmission. A better understanding of the proteins controlling the parasite's life cycle stage transitions is needed for the development of new therapies aimed at treating toxoplasmosis and the transmission of the infection. Genes that regulate the sexual stages need to be turned on and off at the appropriate times, activities that are mediated by specific transcription factors that recruit general machinery to silence or activate gene expression. In this study, we identify a transcription factor called AP2XII-2 as being important for the repression of a subset of sexual stage genes, including a sexual stage-specific AP2 factor (AP2X-10) and a protein (AAH1) required to construct the infectious oocysts expelled from infected cats.

Life cycle and reproduction dynamics of Bangiales in response to environmental stresses.

Red algae of the order Bangiales are notable for exhibiting flexible promotion of sexual and asexual reproductive processes by environmental stresses. This flexibility indicates that a trade-off between vegetative growth and reproduction occurs in response to environmental stresses that influence the timing of phase transition within the life cycle. Despite their high phylogenetic divergence, both filamentous and foliose red alga in the order Bangiales exhibit a haploid-diploid life cycle, with a haploid leafy or filamentous gametophyte (thallus) and a diploid filamentous sporophyte (conchocelis). Unlike haploid-diploid life cycles in other orders, the gametophyte in Bangiales is generated independently of meiosis; the regulation of this generation transition is not fully understood. Based on transcriptome and gene expression analyses, the originally proposed biphasic model for alternation of generations in Bangiales was recently updated to include a third stage. Along with the haploid gametophyte and diploid sporophyte, the triphasic framework recognizes a diploid conchosporophyte-a conchosporangium generated on the conchocelis-phase and previously considered to be part of the sporophyte. In addition to this sexual life cycle, some Bangiales species have an asexual life cycle in which vegetative cells of the thallus develop into haploid asexual spores, which are then released from the thallus to produce clonal thalli. Here, we summarize the current knowledge of the triphasic life cycle and life cycle trade-off in Neopyropia yezoensis and 'Bangia' sp. as model organisms for the Bangiales.

Atypical life cycle does not lead to inbreeding or selfing in parasites despite clonemate accumulation in intermediate hosts.

Many parasites utilize asexual and sexual reproduction and multiple hosts to complete their life cycles. How these taxa avoid inbreeding is an essential question for understanding parasite evolution and ecology. Aquatic trematodes that require multiple host species may benefit from diverse genetic parasite assemblages accumulating within second intermediate hosts prior to sexual reproduction in definitive hosts. However, Cotylurus species are able to utilize the same snail species as first and second intermediate hosts, potentially resulting in the accumulation of genetically identical clones (clonemates) prior to sexual reproduction. In this study, we developed and analysed novel microsatellite loci to determine if clones are accumulating within snail hosts prior to ingestion by bird hosts and the effects this could have on parasite inbreeding. Contrary to previous studies of aquatic trematodes, significantly large numbers of clonemates were present within snails, but full-sibs were not. Genetic structure was present over a relatively small geographical scale despite the use of vagile definitive hosts. Phylogenetic analysis identified the Cotylurus sp. clones as belonging to a single species. Despite the presence of clones within snails, mating between clones/selfing was not common and heterozygosity is maintained within individuals. Potential issues with clones mating may be mitigated by the presence of snails with numerous clones, the consumption of many snails by bird hosts and parasite clone recognition/avoidance. Use of the same host species for multiple life stages may have advantages when parasites are able to avoid inbreeding and the required hosts are common.

Dissection of Besnoitia besnoiti intermediate host life cycle stages: From morphology to gene expression.

Cyst-forming Apicomplexa (CFA) of the Sarcocystidae have a ubiquitous presence as pathogens of humans and farm animals transmitted through the food chain between hosts with few notable exceptions. The defining hallmark of this family of obligate intracellular protists consists of their ability to remain for very long periods as infectious tissue cysts in chronically infected intermediate hosts. Nevertheless, each closely related species has evolved unique strategies to maintain distinct reservoirs on global scales and ensuring efficient transmission to definitive hosts as well as between intermediate hosts. Here, we present an in-depth comparative mRNA expression analysis of the tachyzoite and bradyzoite stages of Besnoitia besnoiti strain Lisbon14 isolated from an infected farm animal based on its annotated genome sequence. The B. besnoiti genome is highly syntenic with that of other CFA and also retains the capacity to encode a large majority of known and inferred factors essential for completing a sexual cycle in a yet unknown definitive host. This work introduces Besnoitia besnoiti as a new model for comparative biology of coccidian tissue cysts which can be readily obtained in high purity. This model provides a framework for addressing fundamental questions about the evolution of tissue cysts and the biology of this pharmacologically intractable infectious parasite stage.

The evolution of reproductive modes and life cycles in amphibians.

Amphibians have undergone important evolutionary transitions in reproductive modes and life-cycles. We compare large-scale macroevolutionary patterns in these transitions across the three major amphibian clades: frogs, salamanders, and caecilians. We analyse matching reproductive and phylogenetic data for 4025 species. We find that having aquatic larvae is ancestral for all three groups and is retained by many extant species (33-44%). The most frequent transitions in each group are to relatively uncommon states: live-bearing in caecilians, paedomorphosis in salamanders, and semi-terrestriality in frogs. All three groups show transitions to more terrestrial reproductive modes, but only in caecilians have these evolved sequentially from most-to-least aquatic. Diversification rates are largely independent of reproductive modes. However, in salamanders direct development accelerates diversification whereas paedomorphosis decreases it. Overall, we find a widespread retention of ancestral modes, decoupling of trait transition rates from patterns of species richness, and the general independence of reproductive modes and diversification.

Comparative genomics of mortal and immortal cnidarians unveils novel keys behind rejuvenation.

Turritopsis dohrnii is the only metazoan able to rejuvenate repeatedly after its medusae reproduce, hinting at biological immortality and challenging our understanding of aging. We present and compare whole-genome assemblies of T. dohrnii and the nonimmortal Turritopsis rubra using automatic and manual annotations, together with the transcriptome of life cycle reversal (LCR) process of T. dohrnii. We have identified variants and expansions of genes associated with replication, DNA repair, telomere maintenance, redox environment, stem cell population, and intercellular communication. Moreover, we have found silencing of polycomb repressive complex 2 targets and activation of pluripotency targets during LCR, which points to these transcription factors as pluripotency inducers in T. dohrnii. Accordingly, we propose these factors as key elements in the ability of T. dohrnii to undergo rejuvenation.