How to resist soil desiccation: Transcriptional changes in a Mediterranean earthworm during aestivation.

Earthworms have a central role in ministering the terrestrial ecosystems and are proving to have an important role in modulating the effects climate change has on soil. Aestivation is a form of dormancy employed by the organisms living in deserts and arid environments, when confronted with prolonged periods of drought. Understanding global metabolic adjustments required for withstanding the harsh conditions of the ever more severe Iberian drought, we performed a global transcriptomic exploration of the endogeic earthworm Carpetania matritensis during aestivation. There were a total of 6352 differentially expressed transcripts in the aestivating group, with 65% being downregulated. Based on GO and KEGG enrichment analyses, downregulated genes seem to be indicative of an overall metabolic depression during aestivation. Indeed we noted a reduction of protein turnover and macromolecule metabolism coupled with suppression of genes involved in digestion. Upregulated genes, namely antioxidant genes and DNA repair genes showed clear signs of abiotic stress caused by ROS generation. Abiotic stress led to transcriptomic changes of genes involved in immune response, mostly affecting the NF-kb signaling pathway as well as changes in apoptotic genes indicating the necessity of investigating these processes in a tissue specific manner. Lastly we uncovered a possible mechanism for water retention by nitrogenous waste accumulation. This study provides the first ever transcriptomic investigation done on aestivating earthworms and as such serves as a general framework for investigation on other earthworm species and other soil invertebrates, which is becoming increasingly important with the current scenario of climate change.

Local adaptation fuels cryptic speciation in terrestrial annelids.

Uncovering the genetic and evolutionary basis of cryptic speciation is a major focus of evolutionary biology. Next Generation Sequencing (NGS) allows the identification of genome-wide local adaptation signatures, but has rarely been applied to cryptic complexes - particularly in the soil milieu - as it is the case with integrative taxonomy. The earthworm genus Carpetania, comprising six previously suggested putative cryptic lineages, is a promising model to study the evolutionary phenomena shaping cryptic speciation in soil-dwelling lineages. Genotyping-By-Sequencing (GBS) was used to provide genome-wide information about genetic variability between 17 populations, and geometric morphometrics analyses of genital chaetae were performed to investigate unexplored cryptic morphological evolution. Genomic analyses revealed the existence of three cryptic species, with half of the previously-identified potential cryptic lineages clustering within them. Local adaptation was detected in more than 800 genes putatively involved in a plethora of biological functions (most notably reproduction, metabolism, immunological response and morphogenesis). Several genes with selection signatures showed shared mutations for each of the cryptic species, and genes under selection were enriched in functions related to regulation of transcription, including SNPs located in UTR regions. Finally, geometric morphometrics approaches partially confirmed the phylogenetic signal of relevant morphological characters such as genital chaetae. Our study therefore unveils that local adaptation and regulatory divergence are key evolutionary forces orchestrating genome evolution in soil fauna.

Gut-derived lipopolysaccharides increase post-prandial oxidative stress via Nox2 activation in patients with impaired fasting glucose tolerance: effect of extra-virgin olive oil.

PURPOSE: Post-prandial phase is characterized by enhanced oxidative stress but the underlying mechanism is unclear. We investigated if gut-derived lipopolysaccharide (LPS) is implicated in this phenomenon and the effect of extra virgin olive oil (EVOO) in patients with impaired fasting glucose (IFG). METHODS: This is a randomized cross-over interventional study including 30 IFG patients, to receive a lunch with or without 10 g of EVOO. Serum LPS, Apo-B48, markers of oxidative stress such as oxidized LDL (oxLDL) and soluble Nox2-derived peptide (sNox2-dp), a marker of nicotinamide-adenine-dinucleotide-phosphate oxidase isoform Nox2 activation, and plasma polyphenols were determined before, 60 and 120 min after lunch. RESULTS: In patients not given EVOO oxidative stress as assessed by sNox2-dp and oxLDL significantly increased at 60 and 120 min concomitantly with an increase of LPS and Apo-B48. In these patients, changes of LPS were correlated with Apo-B48 (Rs = 0.542, p = 0.002) and oxLDL (Rs = 0.463, p = 0.010). At 120 min, LPS (ß - 15.73, p < 0.001), Apo-B48 (ß - 0.14, p = 0.004), sNox2-dp (ß - 5.47, p = 0.030), and oxLDL (ß - 42.80, p < 0.001) significantly differed between the two treatment groups. An inverse correlation was detected between polyphenols and oxLDL (R - 0.474, p < 0.005). In vitro study showed that LPS, at the same concentrations found in the human circulation, up-regulated Nox2-derived oxidative stress via interaction with Toll-like receptor 4. CONCLUSIONS: Post-prandial phase is characterized by an oxidative stress-related inflammation potentially triggered by LPS, a phenomenon mitigated by EVOO administration.

Unravelling the molecular mechanisms of nickel in woodlice.

During the last few years, there has been an alarming increase in the amount of nickel (Ni) being released into the environment, primarily due to its use in the production of stainless steel but also from other sources such as batteries manufacturing and consequent disposal. The established biotic ligand models provide precise estimates for Ni bioavailability, in contrast, studies describing the mechanisms underpinning toxicological effect of Ni are scarce. This study exploits RNA-seq to determine the transcriptomic responses of isopods using Porcellionides pruinosus as an example of a terrestrial metal-resistant woodlouse. Furthermore, the recently proposed model for Ni adverse outcome pathways (Ni-AOP) presents an unprecedented opportunity to fit isopod responses to Ni toxicity and define Porcellionides pruinosus as a metalomic model. Prior to this study, P. pruinosus represented an important environmental sentinel, though lacking genetic/omic data. The reference transcriptome generated here thus represents a major advance and a novel resource. A detailed annotation of the transcripts obtained is presented together with the homology to genes/gene products from Metazoan and Arthropoda phylum, Gene Ontology (GO) classification, clusters of orthologous groups (COG) and assignment to KEGG metabolic pathways. The differential gene expression comparison was determined in response to nickel (Ni) exposure and used to derive the enriched pathways and processes. It revealed a significant impact on ion trafficking and storage, oxidative stress, neurotoxicity, reproduction impairment, genetics and epigenetics. Many of the processes observed support the current Ni-AOP although the data highlights that the current model can be improved by including epigenetic endpoints, which represents key chronic risks under a scenario of Ni toxicity.

Effects on tadpole snail gene expression after exposure to vinclozolin.

The fungicide vinclozolin (Vz) is an endocrine disruptor with known anti-androgenic activity in vertebrates. However, there is a lack of information about the Vz mode of action in invertebrates, although some studies have shown that this compound can produce alterations in different species. Transcriptional activity was analyzed in the freshwater snail Physella acuta in order to elucidate putative cellular processes altered by this chemical during a response. In order to identify potential molecular biomarkers, a de novo transcriptome was generated for this species that constitutes a valuable source for future studies. This data, together with some already available data, permitted the identification of several genes related to detoxification mechanisms (Cyp2u1, Cyp3a7, Cyp4f22, GSTo1, GSTt2, and MRP1), stress response (Hsp20.4, Hsp17, Hsp16.6, and Cu,Zn-SOD), the hormonal system (Estrogen Receptor and Hsp90), apoptosis (Casp3), and copper homeostasis (ATOX1). Using quantitative Real-Time polymerase chain reaction, mRNA levels of these genes were examined in snails exposed to 20 or 200 µg/L Vz for 24 h. The results showed an overall weak response, with downregulation of Hsp20.4 and no statistically significant change for the other genes. These findings suggest that P. acuta can manage the concentrations of Vz found in the environment with no relevant activation of the pathways analyzed, although additional studies are needed for longer exposure times and including other metabolic pathways. The new genes described open the range of processes that can be studied at the molecular level in toxicity tests.

Glucocorticoids impair platelet thromboxane biosynthesis in community-acquired pneumonia.

Previous reports suggest that community-acquired pneumonia (CAP) is associated with an enhanced risk of myocardial infarction (MI) and that enhanced platelet activation may play a role. Aims of this study were to investigate if urinary excretion of 11-dehydro-thromboxane (Tx) B2, a reliable marker of platelet activation in vivo, was elevated in CAP and whether glucocorticoid administration reduced platelet activation. Three-hundred patients hospitalized for CAP were recruited and followed-up until discharge. Within the first 2 days from admission, urinary 11-dehydro-TxB2 and serum levels of methylprednisolone and betamethasone were measured. 11-Dehydro-TxB2 was also measured in a control group of 150 outpatients, matched for age, sex, and comorbidities. Finally, in-vitro studies were performed to assess if glucocorticoids affected platelet activation, at the same range of concentration found in the peripheral circulation of CAP patients treated with glucocorticoids. Compared to controls, CAP patients showed significantly higher levels of 11-dehydro-TxB2 (110 [69-151] vs. 163 [130-225] pg/mg creatinine; p < 0.001). During the in-hospital stay, 31 patients experienced MI (10%). A COX regression analysis showed that 11-dehydro-TxB2 independently predicted MI (p = .005). CAP patients treated with glucocorticoids showed significantly lower levels of 11-dehydro-TxB2 compared to untreated ones (147 [120-201] vs. 176 [143-250] pg/mg creatinine; p < 0.001). In vitro, glucocorticoids-treated platelets showed a dose-dependent decrease of ADP-induced platelet aggregation, TxB2 production, cPLA2 phosphorylation and arachidonic acid release from the platelet membrane. In conclusion, platelet TxB2 is overproduced in CAP patients and may be implicated in MI occurrence. Glucocorticoids reduce platelet release of TxB2 in vitro and urinary excretion of 11-dehydro-TxB2 in vivo and may be a novel tool to decrease platelet activation in this setting.

Oleuropein, a component of extra virgin olive oil, lowers postprandial glycaemia in healthy subjects.

AIMS: Extra virgin olive oil lowers postprandial glycaemia. We investigated if oleuropein, a component of extra virgin olive oil, exerts a similar effect on postprandial glycaemia and the underlying mechanism. METHODS: Twenty healthy subjects were randomly allocated in a cross-over design to 20 mg oleuropein or placebo immediately before lunch. Postprandial glycaemia along with blood insulin, dipeptidyl-peptidase-4 (DPP-4) and glucagon-like peptide-1 and oxidative stress, which included soluble NADPH oxidase-derived peptide activity (sNox2-dp), 8-iso-prostaglandin-2α and platelet p47phox phosphorylation, were analysed before and 2 h after meal. RESULTS: After 2 h, subjects who assumed oleuropein had significantly lower blood glucose, DPP-4 activity and higher insulin and glucagon-like peptide-1 compared to placebo. Furthermore, sNox2-dp, 8-iso-PGF2α and platelet p47phox phosphorylation were significantly lower in oleuropein- compared to placebo-treated subjects. DPP-4 significantly correlated with sNox2-dp [Spearman's rho (Rs) = 0.615; P < 0.001], p47phox phosphorylation (Rs = 0.435; P < 0.05) and 8-iso- prostaglandin-2α (Rs = 0.33; P < 0.05). In vitro study demonstrated that hydroxytyrosol, a metabolite of oleuropein, significantly reduced p47phox phosphorylation and isoprostane formation. CONCLUSIONS: These findings indicate that oleuropein improves postprandial glycaemic profile via hampering Nox2-derived oxidative stress.

Gut-derived endotoxin stimulates factor VIII secretion from endothelial cells. Implications for hypercoagulability in cirrhosis.

BACKGROUND & AIMS: Patients with cirrhosis display enhanced blood levels of factor VIII, which may result in harmful activation of the clotting system; however, the underlying mechanism is unknown. METHODS: We performed a cross-sectional study in patients with cirrhosis (n=61) and matched controls (n=61) comparing blood levels of factor VIII, von Willebrand factor (vWf), lipopolysaccharide (LPS) and positivity for Escherichia coli DNA. Furthermore, we performed an in vitro study to investigate if LPS, in a concentration range similar to that found in the peripheral circulation of cirrhotic patients, was able to elicit factor VIII secretion from human umbilical vein endothelial cells (HUVEC). RESULTS: Patients with cirrhosis displayed higher serum levels of LPS (55.8 [42.2-79.9] vs. 23.0 [7.0-34.0]pg/ml, p<0.001), factor VIII (172.0 [130.0-278.0] vs. 39.0 [26.0-47.0]U/dl, p<0.0001), vWf (265.0 [185.0-366.0] vs. 57.0 [48.0-65.0]U/dl, p<0.001) and positivity for Escherichia coli DNA (88% vs. 3%, p<0.001, n=34) compared to controls. Serum LPS correlated significantly with factor VIII (r=0.80, p<0.001) and vWf (r=0.63, p<0.001). Only LPS (beta-coefficient=0.70, p<0.0001) independently predicted factor VIII levels. The in vitro study showed that LPS provoked factor VIII and vWf release from HUVEC via formation and secretion of Weibel-Palade bodies, a phenomenon blunted by pre-treating HUVEC with an inhibitor of Toll-like receptor 4. CONCLUSIONS: The study provides the first evidence that LPS derived from gut microbiota increases the systemic levels of factor VIII via stimulating its release by endothelial cells. Lay summary: Cirrhosis is associated with thrombosis in portal and systemic circulation. Enhanced levels of factor VIII have been suggested to play a role but the underlying mechanism is still unclear. Here we show that patients with cirrhosis display a concomitant increase of factor VIII and lipopolysaccharide (LPS) from Escherichia coli and suggest that LPS contributes to the release of factor VIII from endothelial cells.

Pinpointing cryptic borders: Fine-scale phylogeography and genetic landscape analysis of the Hormogaster elisae complex (Oligochaeta, Hormogastridae).

Spatial and temporal aspects of the evolution of cryptic species complexes have received less attention than species delimitation within them. The phylogeography of the cryptic complex Hormogaster elisae (Oligochaeta, Hormogastridae) lacks knowledge on several aspects, including the small-scale distribution of its lineages or the palaeogeographic context of their diversification. To shed light on these topics, a dense specimen collection was performed in the center of the Iberian Peninsula - resulting in 28 new H. elisae collecting points, some of them as close as 760m from each other- for a higher resolution of the distribution of the cryptic lineages and the relationships between the populations. Seven molecular regions were amplified: mitochondrial subunit 1 of cytochrome c oxidase (COI), 16S rRNA and tRNA Leu, Ala, and Ser (16S t-RNAs), one nuclear ribosomal gene (a fragment of 28S rRNA) and one nuclear protein-encoding gene (histone H3) in order to infer their phylogenetic relationships. Different representation methods of the pairwise divergence in the cytochrome oxidase I sequence (heatmap and genetic landscape graphs) were used to visualize the genetic structure of H. elisae. A nested approach sensu Mairal et al. (2015) (connecting the evolutionary rates of two datasets of different taxonomic coverage) was used to obtain one approximation to a time-calibrated phylogenetic tree based on external Clitellata fossils and a wide molecular dataset. Our results indicate that limited active dispersal ability and ecological or biotic barriers could explain the isolation of the different cryptic lineages, which never co-occur. Rare events of long distance dispersal through hydrochory appear as one of the possible causes of range expansion.

Articulating "Archiannelids": Phylogenomics and Annelid Relationships, with Emphasis on Meiofaunal Taxa.

Annelid disparity has resulted in morphological-based classifications that disagree with phylogenies based on Sanger sequencing and phylogenomic analyses. However, the data used for the latter studies came from various sources and technologies, involved poorly occupied matrices and lacked key lineages. Here, we generated a new Illumina-based data set to address annelid relationships from a fresh perspective, independent from previously generated data and with nearly fully occupied matrices. Our sampling reflects the span of annelid diversity, including two symbiotic annelid groups (Myzostomida and Spinther) and five meiofaunal groups once referred to as part of Archiannelida (three from Protodrilida, plus Dinophilus and Polygordius). As well as the placement of these unusual annelids, we sought to address the overall phylogeny of Annelida, and provide a new perspective for naming of major clades. Our results largely corroborate the phylogenomic results of Weigert et al. (2014; Illuminating the base of the annelid tree using transcriptomics. Mol Biol Evol. 31:1391-1401), with "Magelona + Owenia" and Chaetopteridae forming a grade with respect to all other annelids. Echiura and Sipuncula are supported as being annelid groups, with Sipuncula closest to amphinomids as sister group to Sedentaria and Errantia. We recovered the three Protodrilida terminals as sister clade to Phyllodocida and Eunicida (=clade Aciculata). We therefore place Protodrilida as part of Errantia. Polygordius was found to be sister group to the scaleworm terminal and the possibility that it is a simplified scaleworm clade, as has been shown for the former family Pisionidae, is discussed. Our results were equivocal with respect to Dinophilus, Myzostomida, and Spinther possibly owing to confounding long-branch effects.

Diversification patterns in cosmopolitan earthworms: similar mode but different tempo.

Comparative phylogeography of widespread species that span the same geographic areas can elucidate the influence of historical events on current patterns of biodiversity, identify patterns of co-vicariance, and therefore aid the understanding of general evolutionary processes. Soil-dwelling animals present characteristics that make them suitable for testing the effect of the palaeogeographical events on their distribution and diversification, such as their low vagility and population structure. In this study, we shed light on the spatial lineage diversification and cladogenesis of two widely-distributed cosmopolitan and invasive earthworms (Aporrectodea rosea and A. trapezoides) in their putative ancestral area of origin, the Western Palearctic, and a few populations in North America. Molecular analyses were conducted on mitochondrial and nuclear markers from 220 (A. rosea) and 198 (A. trapezoides) individuals collected in 56 and 57 localities, respectively. We compared the lineage diversification pattern, genetic variability and cladogenesis in both species. Our findings showed that both species underwent a similar diversification from the Western Mediterranean plates to (i) Northern Europe and (ii) the Iberian Peninsula, establishing their two main lineages. Their diversification was in concordance with the main palaeogeographical events in the Iberian Peninsula and Western Mediterranean, followed by a later colonization of North America from individuals derived exclusively from the Eurosiberian lineage. Their diversification occurred at different times, with the diversification of A. rosea being potentially more ancient. Cladogenesis in both species seems to have been modelled only by the Mediterranean plate shifts, ignoring historical climatic oscillations such as the Messinian salinity crisis. Their high genetic variability, strong population structure, lack of gene flow and stepping-stone-like cladogenesis suggest the existence of different cryptic lineages. Our results may indicate a recurrent event in invasive earthworms within their ancestral distribution areas in the Western Palearctic.

Phylogenomic analyses of a Mediterranean earthworm family (Annelida: Hormogastridae).

Earthworm taxonomy and evolutionary biology remain a challenge because of their scarce distinct morphological characters of taxonomic value, the morphological convergence by adaptation to the uniformity of the soil where they inhabit, and their high plasticity when challenged with stressful or new environmental conditions. Here we present a phylogenomic study of the family Hormogastridae, representing also the first piece of work of this type within earthworms. We included seven transcriptomes of the group representing the main lineages as previously-described, analysed in a final matrix that includes twelve earthworms and eleven outgroups. While there is a high degree of gene conflict in the generated trees that obscure some of the internal relationships, the origin of the family is well resolved: the hormogastrid Hemigastrodrilus appears as the most ancestral group, followed by the ailoscolecid Ailoscolex, therefore rejecting the validity of the family Ailoscolecidae. Our results place the origin of hormogastrids in Southern France, as previously hypothesised.

Pheromone evolution, reproductive genes, and comparative transcriptomics in mediterranean earthworms (annelida, oligochaeta, hormogastridae).

Animals inhabiting cryptic environments are often subjected to morphological stasis due to the lack of obvious agents driving selection, and hence chemical cues may be important drivers of sexual selection and individual recognition. Here, we provide a comparative analysis of de novo-assembled transcriptomes in two Mediterranean earthworm species with the objective to detect pheromone proteins and other reproductive genes that could be involved in cryptic speciation processes, as recently characterized in other earthworm species. cDNA libraries of unspecific tissue of Hormogaster samnitica and three different tissues of H. elisae were sequenced in an Illumina Genome Analyzer II or Hi-Seq. Two pheromones, Attractin and Temptin were detected in all tissue samples and both species. Attractin resulted in a reliable marker for phylogenetic inference. Temptin contained multiple paralogs and was slightly overexpressed in the digestive tissue, suggesting that these pheromones could be released with the casts. Genes involved in sexual determination and fertilization were highly expressed in reproductive tissue. This is thus the first detailed analysis of the molecular machinery of sexual reproduction in earthworms.

Surviving in a multistressor world: Gene expression changes in earthworms exposed to heat, desiccation, and chemicals.

An investigation of the effects of anthropogenic stress on terrestrial ecosystems is urgently needed. In this work, we explored how exposure to heat, desiccation, and chemical stress alters the expression of genes that encode heat shock proteins (HSPs), an enzyme that responds to oxidative stress (CAT), hypoxia-related proteins (HIF1 and HYOU), and a DNA repair-related protein (PARP1) in the earthworm Eisenia fetida. Exposure to heat (31°C) for 24 h upregulated HSPs and hypoxia-related genes, suggesting possible acquired thermotolerance. Desiccation showed a similar expression profile; however, the HSP response was activated to a lesser extent. Heat and desiccation activated the small HSP at 24 h, suggesting that they may play a role in adaptation. Simultaneous exposure to endosulfan and temperature for 7 h upregulated all of the evaluated genes, implicating a coordinated response involving multiple biological processes to ensure survival and acclimation. These results highlight the relevance of multistress analysis in terrestrial invertebrates.

Terrestrial Zen master: Transcriptomics in long-term aestivation and arousal of Mediterranean earthworms.

Earthworms have a crucial role in the maintenance of the biotic and abiotic soil properties, which is important for the biodiversity and productivity of terrestrial ecosystems, especially in the current scenario of climate change. Aestivation is a form of dormancy witnessed in organisms living in deserts or semiarid environments such as the ones found in the central part of the Iberian Peninsula. This work employs next-generation sequencing techniques to explore the changes in gene expression of different aestivation times (1 month and 1 year) as well as changes in gene expression upon arousal. Not surprisingly, the more the aestivation persisted the higher levels of gene downregulation were observed. Conversely, upon arousal, a quick recovery of the levels of gene expression were noted, comparable to the control. Transcriptional changes related to immune responses coming predominantly from abiotic stressors in aestivating earthworms and from biotic stressors in aroused earthworms triggered regulation of the cell fate via apoptosis. Long-term aestivation seemed to be enabled by remodeling of the extracellular matrix, activity of DNA repair mechanisms, and inhibitory neurotransmitters, which could also play a role in lifespan increase. Arousal from 1-month aestivation was on the other hand, characterized by regulation of the cell division cycle. Since aestivation is considered as an unfavorable metabolic state, aroused earthworms probably go through a damage removal process and a subsequent reparation process. This study provides the first transcriptomic investigation done on earthworms in such long aestivation times as well as arousal demonstrating the resilience and adaptability of Carpetania matritensis.

Contribution to the knowledge of the genus Boucheona (Oligochaeta, Hormogastridae) in France with a newly described species and a redescribed species.

Hormogastrid earthworms are found in the diversity hotspot of the Franco-Iberian domain, together with the better-known family Lumbricidae. Integrative systematics (the combination of morphological, molecular and ecological data) have increased our knowledge of the diversity and evolutionary history of these earthworms, highlighting unresolved taxonomic conflicts. One example of a species group in need of integrative taxonomic revision is the genus Boucheona in France. In this work, we analyzed their diversity using previously published data together with additional data obtained from recently sampled localities. Molecular data including DNA barcodes and additional markers enabled us to reconstruct Bayesian and time-calibrated phylogenies to discuss the evolutionary relationships among the different taxa, and to propose hypotheses regarding their biogeographical history. Based on our results, four species of Boucheona are present in Southern France, including two new taxa. Morphological distinctness and molecular phylogenetics results supported the status of four populations as the newly described Boucheona corbierensis sp. nov., as well as the status of "Hormogaster pretiosa var. nigra" as an independent species, redescribed as Boucheona tenebrae sp. nov. These results provide a new perspective of the importance of the genus Boucheona in southern France, as the possible evolutionary origin of a clade of giant anecic earthworms with unknown (but probably remarkable) impact on ecosystem functioning across their range.

Ovary micromorphology in hormogastrid earthworms with a particular emphasis on the organization of the germline cysts.

There is a gap in our knowledge of microorganization and the functioning of ovaries in earthworms (Crassiclitellata) and allied taxa. Recent analyses of ovaries in microdriles and leech-like taxa revealed that they are composed of syncytial germline cysts accompanied by somatic cells. Although the pattern of cyst organization is conserved across Clitellata - each cell is connected via one intercellular bridge (ring canal) to the central and anuclear cytoplasmic mass termed the cytophore - this system shows high evolutionary plasticity. In Crassiclitellata, only the gross morphology of ovaries and their segmental localization is well known, whereas ultrastructural data are limited to lumbricids like Dendrobaena veneta. Here we present the first report about ovarian histology and ultrastructure in Hormogastridae, a small family of earthworms inhabiting the western parts of the Mediterranean sea basin. We analyzed three species from three different genera and showed that the pattern of ovary organization is the same within this taxon. Ovaries are cone-like, with a broad part connected to the septum and a narrow distal end forming an egg string. Ovaries are composed of numerous cysts uniting a small number of cells, eight in Carpetania matritensis. There is a gradient of cysts development along the long ovary axis, and three zones can be distinguished. In zone I, cysts develop in complete synchrony and unite oogonia and early meiotic cells (till diplotene). Then (zone II), the synchrony is lost, and one cell (prospective oocyte) grows faster than the rest (prospective nurse cells). In zone III, oocytes pass the growth phase and gather nutrients; at this time, their contact with the cytophore is lost. Nurse cells grow slightly, eventually die via apoptosis, and are removed by coelomocytes. The most characteristic feature of hormogastrid germ cysts is the inconspicuous cytophore in the form of thread-like thin cytoplasmic strands (reticular cytophore). We found that the ovary organization in studied hormogastrids is very similar to that described for D. veneta and propose the term "Dendrobaena" type of ovaries. We expect the same microorganization of ovaries will be found in other hormogastrids and lumbricids.

mRNA COVID-19 Vaccination Does Not Exacerbate Symptoms or Trigger Neural Antibody Responses in Multiple Sclerosis.

BACKGROUND AND OBJECTIVE: In people with multiple sclerosis (pwMS), concern for potential disease exacerbation or triggering of other autoimmune disorders contributes to vaccine hesitancy. We assessed the humoral and T-cell responses to SARS-CoV-2 after mRNA vaccination, changes in disease activity, and development of antibodies against central or peripheral nervous system antigens. METHODS: This was a prospective 1-year longitudinal observational study of pwMS and a control group of patients with other inflammatory neurologic disorders (OIND) who received an mRNA vaccine. Blood samples were obtained before the first dose (T1), 1 month after the first dose (T2), 1 month after the second dose (T3), and 6 (T4), 9 (T5), and 12 (T6) months after the first dose. Patients were assessed for the immune-specific response, annualized relapse rate (ARR), and antibodies to onconeuronal, neural surface, glial, ganglioside, and nodo-paranodal antigens. RESULTS: Among 454 patients studied, 390 had MS (22 adolescents) and 64 OIND; the mean (SD) age was 44 (14) years; 315 (69%) were female; and 392 (87%) were on disease-modifying therapies. Antibodies to the receptor-binding domain were detected in 367 (86%) patients at T3 and 276 (83%) at T4. After a third dose, only 13 (22%) of 60 seronegative patients seroconverted, and 255 (92%) remained seropositive at T6. Cellular responses were present in 381 (93%) patients at T3 and in 235 (91%) patients at T6 including all those receiving anti-CD20 therapies and in 79% of patients receiving fingolimod. At T3 (429 patients) or T6 (395 patients), none of the patients had developed CNS autoantibodies. Seven patients had neural antibodies that were already present before immunization (3 adult patients with MS had MOG-IgG, 2 with MG and 1 with MS had neuronal cell surface antibodies [unknown antigen], and 1 with MS had myelin antibody reactivity [unknown antigen]. Similarly, no antibodies against PNS antigens were identified at T3 (427 patients). ARR was lower in MS and not significantly different in patients with OIND. Although 182 (40%) patients developed SARS-CoV-2 infection, no cases of severe COVID-19 or serious adverse events occurred. DISCUSSION: In this study, mRNA COVID-19 vaccination was safe and did not exacerbate the autoimmune disease nor triggered neural autoantibodies or immune-mediated neurologic disorders. The outcome of patients who developed SARS-CoV-2 infection was favorable.

Appearances can be deceptive: different diversification patterns within a group of Mediterranean earthworms (Oligochaeta, Hormogastridae).

Many recent studies on invertebrates have shown how morphology not always captures the true diversity of taxa, with cryptic speciation often being discussed in this context. Here, we show how diversification patterns can be very different in two clades of closely related earthworms in the genus Hormogaster stressing the risk of using nonspecific substitution rate values across taxa. On the one hand, the Hormogaster elisae species complex, endemic to the central Iberian Peninsula, shows morphological stasis. On the other hand, a clade of Hormogaster from the NE Iberian Peninsula shows an enormous morphological variability, with 15 described morphospecies. The H. elisae complex, however, evolves faster genetically, and this could be explained by the harsher environmental conditions to which it is confined-as detected in this study, that is, sandier and slightly poorer soils with lower pH values than those of the other species in the family. These extreme conditions could be at the same time limiting morphological evolution and thus be responsible for the observed morphological stasis in this clade. Contrarily, Hormogaster species from the NE Iberian Peninsula, although still inhabiting harsher milieu than other earthworm groups, have had the opportunity to evolve into a greater morphological disparity. An attempt to delimit species within this group following the recently proposed general mixed Yule-coalescent method showed a higher number of entities than expected under the morphospecies concept, most probably due to the low vagility of these animals, which considerably limits gene flow between distant conspecific populations, but also because of the decoupling between morphological and genetic evolution in the H. elisae complex.