Excito-repellent and Pesticide-Likeness Properties of Essential Oils on Carpophilus dimidiatus (Fabricius) (Nitidulidae) and Oryzaephilus mercator (L.) (Silvanidae).

Carpophilus dimidiatus (Fabricius) (Nitidulidae) and Oryzaephilus mercator (L.) (Silvanidae) are insect pests that cause severe damage in important walnut growing regions in the northwest of Argentina. The current management approaches for these pests involve the use of unsafe phosphorus pesticides whose overuse have led to farmworker poisoning, pest resistance issues, and environmental contamination. Plant extracts, particularly essential oils, are an alternative source of insect control agents. Excito-repellent essential oils can be used to develop ecofriendly tools for managing the pest population without affecting quality and visual appearance of the stored walnuts. Laboratory studies were conducted to assess the excito-repellent effects of C. dimidiatus and O. mercator of 12 essential oils derived from aromatic plants used as food additives and traditional medicine in Argentina: Aloysia citrodora (AC), Aloysia gratissima (AG), Aloysia gratissima var. Gratissima (AGG), Blepharocalyx salicipholius (BS), Hyptis mutabilis (HM), Lippia junelliana (LJ), Lippia turbinata (LT), Mentha x piperita (MP), Minthostachys mollis (MM), Minthostachys verticillata (MV), Origanum vulgare(OV), and Rosmarinus officinalis (RO). The most bioactive EOs (ERijk ≥ 70%) were Aloysia gratissima var. Gratissima (AGG), Minthostachys verticillata, and Lippia junelliana. Their bioactivity profile and chemical space, characterized from GC-MS measures, Generalized Estimating Equations, and Hierarchical Cluster Analysis, revealed that they are mixtures of very functionalized molecules with physicochemical properties similar to those of insecticides with low residual property that enter the insect body through the respiratory system by inhalation. The AGG, MV, and LJ oils are promising as protective agents of walnut products. In our laboratory, studies of their formulations for use in integrated pest management programs are still ongoing.

Insertion Torque and Resonance Frequency Analysis in Tapered and Parallel Dental Implants.

Primary implant stability (PIS) is known to vary with recipient bone mass and density, dental implant design and surgical technique. The objective of this preliminary study was to compare rotational and lateral PIS of same-coronal-diameter conical and parallel implants, using insertion torque recorded with a dental implant motor set and implant stability quotient obtained from resonance frequency analysis (performed with both Osstell and Penguin systems) as measures of rotational and lateral stability, respectively. Additionally, the relationship between PIS and alveolar ridge width (ARW) was explored in both implant types. Sixty dental implants (30 tapered and 30 parallel) were randomly placed with a split-mouth design in 17 patients. Bone density and ARW were estimated from cone beam computed tomography images taken with radiological-surgical templates. Density and width values were similar in the 2 groups (P > .05). Implant coronal diameters were 3.75 mm in all cases, while consistent with the manufacturer's recommendations, final drill bit diameters used were 3.25 and 3.4 mm for parallel and tapered implants, respectively. Insertion torque was higher (P < .05) with parallel implants, but between-group differences in implant stability quotient were not significant (P > .05). In tapered implants, insertion torque was inversely correlated with ARW (P < .001). Notably, significant differences were observed between resonance frequency analysis values from Osstell and Penguin systems (P < .001). In conclusion, future studies should explore how PIS may be influenced by final drill bit size regardless of implant design and potential limits on the effectiveness of tapered implants to achieve good stability in thick low-density bone.

Effectiveness, Toxicity, and Survival Predictors of Regorafenib in Metastatic Colorectal Cancer: A Multicenter Study of Routinely Collected Data.

OBJECTIVES: To assess the effectiveness and toxicity of regorafenib in patients with metastatic colorectal cancer (mCRC) in routine clinical practice, as well as predictive factors of effectiveness. METHODS: This was a retrospective multicenter study in patients with mCRC who received regorafenib from November 2013 to May 2020. Effectiveness was evaluated by overall survival (OS) and progression-free survival (PFS) using the Kaplan-Meier method. Cox regression was performed to determine survival predictors. RESULTS: Ninety patients were enrolled (median age, 64.3 years). Fifty-two patients (57.8%) were male, and 57 (63.3%) had an ECOG performance status (PS) of 0 to 1. Median follow-up was 2.80 months. Median OS was 8.03 months (95% CI, 5.90-10.17), and median PFS was 2.90 months (95% CI, 2.59-3.21). Eighty-eight patients (97.8%) experienced drug-related adverse events. The most frequent were fatigue in 66 patients (73.3%), followed by palmar-plantar erythrodysesthesia in 40 (44.4%). Low liver tumor burden score (LTBS) and good ECOG PS were independent OS predictive factors. CONCLUSIONS: Patients taking regorafenib had OS and PFS rates similar to those reported in previous randomized trials; the agent had a poor toxicity profile. We identified low LTBS and good ECOG PS as possible predictive factors of better OS, useful in selecting patients with mCRC who might benefit from regorafenib.

Medicago truncatula Yellow Stripe-Like7 encodes a peptide transporter participating in symbiotic nitrogen fixation.

Yellow Stripe-Like (YSL) proteins are a family of plant transporters that are typically involved in transition metal homeostasis. Three of the four YSL clades (I, II and IV) transport metals complexed with the non-proteinogenic amino acid nicotianamine or its derivatives. No such capability has been shown for any member of clade III, but the link between these YSLs and metal homeostasis could be masked by functional redundancy. We studied the role of the clade III YSL protein MtSYL7 in Medicago truncatula nodules. MtYSL7, which encodes a plasma membrane-bound protein, is mainly expressed in the pericycle and cortex cells of the root nodules. Yeast complementation assays revealed that MtSYL7 can transport short peptides. M. truncatula transposon insertion mutants with decreased expression of MtYSL7 had lower nitrogen fixation rates and showed reduced plant growth whether grown in symbiosis with rhizobia or not. YSL7 mutants accumulated more copper and iron in the nodules, which is likely to result from the increased expression of iron uptake and delivery genes in roots. Taken together, these data suggest that MtYSL7 plays an important role in the transition metal homeostasis of nodules and symbiotic nitrogen fixation.

Treatment of classical Hodgkin lymphoma in young adults aged 18-30 years with a modified paediatric Hodgkin lymphoma protocol. Results of a multicentre phase II clinical trial (CRUK/08/012).

This phase II trial was designed to determine the safety and efficacy of a modified paediatric risk-stratified protocol in young adults (18-30 years) with classical Hodgkin Lymphoma. The primary end-point was neurotoxicity rate. The incidence of grade 3 neurotoxicity was 11% (80% CI, 5-19%); a true rate of neuropathy of >15% cannot be excluded. Neuropathy and associated deterioration in quality of life was largely reversible. The overall response rate was 100% with 40% complete remission (CR) rate. Twelve months disease-free survival (DFS) was 91%. We demonstrate that a risk-stratified paediatric combined modality treatment approach can be delivered to young adults without significant irreversible neuropathy.

Self-setup of home respiratory polygraphy for the diagnosis of sleep apnea syndrome: cost-efficiency study.

PURPOSE: To evaluate the differences in reliability and costs of home respiratory polygraphy (HRP) when installed by the patient and by a nurse, in order to determine the factors affecting and to consider the possible generalization of self-setup procedure. Several HRP devices have been validated for obstructive sleep apnea (OSA) diagnosis but convenience of a nurse intervention in HRP installation has been scarcely studied. METHODS: This is a prospective and interventional study. About 301 participants were assigned to 2 groups: self-setup and nurse intervention. Sleep study, questionnaires, and diagnostic procedures were performed following the clinical practice in 2016. Signals were considered lost above 3 min, and success of the test was established according to guidelines. Costs were calculated according to a previous multicenter study. RESULTS: Both groups (self-setup and nurse intervention) resulted homogeneous in age, gender, BMI, and final diagnosis of OSA. Signal losses during the test were similar in both groups. Slightly higher percentage of unsuccessful tests were obtained in the self-setup procedure (5.3 vs 2.0%, p = 0.121). The costs were similar (107 vs 105 €) in the self-setup group as compared to the nurse setup group. CONCLUSIONS: The setup of HRP by either the patient or nurse had similar costs and data acquisition. Both installation procedures of HRP were similar regarding test reliability and costs. Main findings are that self-installation by the patient could be similarly reliable and economic as installation by a nurse, as far as consensus guidelines are followed. This study demonstrates that self-setup of HRP is a potentially viable option for the diagnosis of OSA.

MtMOT1.2 is responsible for molybdate supply to Medicago truncatula nodules.

Symbiotic nitrogen fixation in legume root nodules requires a steady supply of molybdenum for synthesis of the iron-molybdenum cofactor of nitrogenase. This nutrient has to be provided by the host plant from the soil, crossing several symplastically disconnected compartments through molybdate transporters, including members of the MOT1 family. Medicago truncatula Molybdate Transporter (MtMOT) 1.2 is a Medicago truncatula MOT1 family member located in the endodermal cells in roots and nodules. Immunolocalization of a tagged MtMOT1.2 indicates that it is associated to the plasma membrane and to intracellular membrane systems, where it would be transporting molybdate towards the cytosol, as indicated in yeast transport assays. Loss-of-function mot1.2-1 mutant showed reduced growth compared with wild-type plants when nitrogen fixation was required but not when nitrogen was provided as nitrate. While no effect on molybdenum-dependent nitrate reductase activity was observed, nitrogenase activity was severely affected, explaining the observed difference of growth depending on nitrogen source. This phenotype was the result of molybdate not reaching the nitrogen-fixing nodules, since genetic complementation with a wild-type MtMOT1.2 gene or molybdate-fortification of the nutrient solution, both restored wild-type levels of growth and nitrogenase activity. These results support a model in which MtMOT1.2 would mediate molybdate delivery by the vasculature into the nodules.

Medicago truncatula Molybdate Transporter type 1 (MtMOT1.3) is a plasma membrane molybdenum transporter required for nitrogenase activity in root nodules under molybdenum deficiency.

Molybdenum, as a component of the iron-molybdenum cofactor of nitrogenase, is essential for symbiotic nitrogen fixation. This nutrient has to be provided by the host plant through molybdate transporters. Members of the molybdate transporter family Molybdate Transporter type 1 (MOT1) were identified in the model legume Medicago truncatula and their expression in nodules was determined. Yeast toxicity assays, confocal microscopy, and phenotypical characterization of a Transposable Element from Nicotiana tabacum (Tnt1) insertional mutant line were carried out in the one M. truncatula MOT1 family member specifically expressed in nodules. Among the five MOT1 members present in the M. truncatula genome, MtMOT1.3 is the only one uniquely expressed in nodules. MtMOT1.3 shows molybdate transport capabilities when expressed in yeast. Immunolocalization studies revealed that MtMOT1.3 is located in the plasma membrane of nodule cells. A mot1.3-1 knockout mutant showed impaired growth concomitant with a reduction of nitrogenase activity. This phenotype was rescued by increasing molybdate concentrations in the nutritive solution, or upon addition of an assimilable nitrogen source. Furthermore, mot1.3-1 plants transformed with a functional copy of MtMOT1.3 showed a wild-type-like phenotype. These data are consistent with a model in which MtMOT1.3 is responsible for introducing molybdate into nodule cells, which is later used to synthesize functional nitrogenase.

Sugary secretions of wasp galls: a want-to-be extrafloral nectar?

Background and Aims: The most widespread form of protective mutualisms is represented by plants bearing extrafloral nectaries (EFNs) that attract ants and other arthropods for indirect defence. Another, but less common, form of sugary secretion for indirect defence occurs in galls induced by cynipid wasps. Until now, such galls have been reported only for cynipid wasps that infest oak trees in the northern hemisphere. This study provides the first evidence of galls that exude sugary secretions in the southern hemisphere and asks whether they can be considered as analogues of plants' EFNs. Methods: The ecology and anatomy of galls and the chemical composition of the secretion were investigated in north-western Argentina, in natural populations of the host trees Prosopis chilensis and P. flexuosa . To examine whether ants protect the galls from natural enemies, ant exclusion experiments were conducted in the field. Key Results: The galls produce large amounts of sucrose-rich, nectar-like secretions. No typical nectary and sub-nectary parenchymatic tissues or secretory trichomes can be observed; instead there is a dense vascularization with phloem elements reaching the gall periphery. At least six species of ants, but also vespid wasps, Diptera and Coleoptera, consumed the gall secretions. The ant exclusion experiment showed that when ants tended galls, no differences were found in the rate of successful emergence of gall wasps or in the rate of parasitism and inquiline infestation compared with ant-excluded galls. Conclusions: The gall sugary secretion is not analogous to extrafloral nectar because no nectar-producing structure is associated with it, but is functionally equivalent to arthropod honeydew because it provides indirect defence to the plant parasite. As in other facultative mutualisms mediated by sugary secretions, the gall secretion triggers a complex multispecies interaction, in which the outcome of individual pair-wise interactions depends on the ecological context in which they take place.

4 Gy versus 24 Gy radiotherapy for patients with indolent lymphoma (FORT): a randomised phase 3 non-inferiority trial.

BACKGROUND: Follicular lymphoma has been shown to be highly radiosensitive with responses to doses as low as 4 Gy in two fractions. This trial was designed to explore the dose response for follicular lymphoma comparing 4 Gy in two fractions with 24 Gy in 12 fractions METHODS: FORT is a prospective randomised, unblinded, phase 3 non-inferiority study comparing radiotherapy given as 4 Gy in two fractions with a standard dose of 24 Gy in 12 fractions. Entry criteria included all patients aged over 18 years, having local radiotherapy for radical or palliative local control, with follicular lymphoma or marginal zone lymphoma, who had received no previous treatment for at least 1 month before. The primary outcome was time to local progression analysed on an intention-to-treat basis. Randomisation was centralised through the Cancer Research UK and University College London Cancer Trials Centre. Radiotherapy target sites were randomised (1:1) with minimisation stratified by histology (follicular lymphoma vs marginal zone lymphoma), treatment intent (palliative or curative) and centre. This trial is registered with ClinicalTrials.gov number, NCT00310167. FINDINGS: 299 sites were randomly assigned to 24 Gy and 315 sites to 4 Gy between April 7, 2006, and June 8, 2011, at 43 centres in the UK. After a median follow-up of 26 months (range 0·39-75·4), 91 local progressions had been recorded (21 in the 24 Gy group and 70 in the 4 Gy group). Time to local progression with 4 Gy was not non-inferior to 24 Gy (hazard ratio 3·42, 95% CI 2·09-5·55, p<0·0001). Eight (3%) of 282 patients in the 24 Gy group and four (1%) of 300 in the 4 Gy group had acute grade 3-4 toxic effects. Four (1%) patients in the 24 Gy group and four (1%) patients in the 4 Gy group had late toxic effects. Mucositis was the most common event in the 24 Gy group (two patients with acute mucositis and two with late mucositis; all grade 3) and was not reported in the 4 Gy group. The most common acute effect was pain at the site of irradiation (two patients in the 4 Gy group, one patient in the 24 Gy group; all grade 3), and the most common late effect was fatigue (two patients in the 4 Gy group, one patient in the 24 Gy group; all grade 3). INTERPRETATION: 24 Gy in 12 fractions is the more effective radiation schedule for indolent lymphoma and should be regarded as the standard of care. However, 4 Gy remains a useful alternative for palliative treatment. FUNDING: Cancer Research UK.

Revision of the ocellatus species-group of the genus Plumarius Philippi 1873 (Hymenoptera, Chrysidoidea, Plumariidae), with description of three new species from Argentina.

This contribution presents the study of species-group of Plumarius Philippi 1873, as the result of a survey of the morphological variation in males. The group is characterized by the large ocelli and transverse head. It consists of the following three new species: P. culminatus Diez, Fidalgo & Roig-Alsina, sp. nov., P. ocellatus Diez, Fidalgo & Roig-Alsina, sp. nov., and P. stangei Diez, Fidalgo & Roig-Alsina, sp. nov. A characterization of the group, descriptions, and illustrations are provided.

Optimised conformal total body irradiation: a heterogeneous practice, so where next?

The use of volumetric arc therapy and inverse planning has been in routine use in radiotherapy for two decades. However, use in total body irradiation (TBI) has been more recent and few guidelines exist as to how to plan or verify. This has led to heterogeneous approaches. The goal of this review is to provide an overview of current advanced planning and dosimetry verification protocols used in optimised conformal TBI as a basis for investigating the need for greater standardisation in TBI.

ALL-RIC trial protocol: a comparison of reduced dose total body irradiation (TBI) and cyclophosphamide with fludarabine and melphalan reduced intensity conditioning in adults with acute lymphoblastic leukaemia (ALL) in complete remission.

INTRODUCTION: The usage of a T-cell depleted, reduced intensity conditioning (RIC) approach to haematopoietic cell transplantation (HCT) in adult patients with acute lymphoblastic leukaemia (ALL) over 40 years of age and in first complete remission (CR) has resulted in encouraging rates of event-free and overall survival in a population of adults with high risk disease. However, relapse rates remain high-with disease progression being the major cause of treatment failure. Using different, more powerful conditioning approaches is the logical next step in examining the role of RIC allogeneic HCT in adult ALL. METHODS AND ANALYSIS: The ALL-RIC trial is a two-arm, phase II, multicentre, randomised clinical trial in adult patients with ALL in first or second CR, who are undergoing allogeneic HCT. Comparison of a novel RIC transplant conditioning regimen using reduced-dose total body irradiation (TBI), cyclophosphamide and alemtuzumab, is made against a standardised RIC approach using fludarabine, melphalan and alemtuzumab. The primary outcome of the study is disease-free survival at 3 years, defined as time from randomisation to the first of either relapse or death from any cause. Patients who are still alive and progression-free at the end of the trial will be censored at their last date known to be alive. Secondary outcomes include overall survival and non-relapse mortality. ETHICS AND DISSEMINATION: The protocol was approved by the East Midlands-Leicester Central Research Ethics committee (18/EM/0112). Initial approval was received on 12 June 2018. Current protocol version (V.6.0) approval obtained on 18 November 2019. The Medicines and Healthcare products Regulatory Agency (MHRA) also approved all protocol versions. The results of this trial will be disseminated through national and international presentations and peer-reviewed publications. TRIAL REGISTRATION NUMBER: EudraCT Number: 2017-004800-23.ISRCTN99927695.

CryoEM reveals that ribosomes in microsporidian spores are locked in a dimeric hibernating state.

Translational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of electron cryo-tomography and single-particle electron cryo-microscopy. We show that microsporidian spores contain hibernating ribosomes that are locked in a dimeric (100S) state, which is formed by a unique dimerization mechanism involving the beak region. The ribosomes within the dimer are fully assembled, suggesting that they are ready to be activated once the host cell is invaded. This study provides structural evidence for dimerization acting as a mechanism for ribosomal hibernation in microsporidia, and therefore demonstrates that eukaryotes utilize this mechanism in translational control.

SEAFARER - A new concept for validating radiotherapy patient specific QA for clinical trials and clinical practice.

BACKGROUND: The quality of radiotherapy delivery has been shown to significantly impact clinical outcomes including patient survival. To identify errors, institutions perform Patient Specific Quality Assurance (PSQA) assessing each individual radiotherapy plan prior to starting patient treatments. Externally administered Dosimetry Audits have found problems despite institutions passing their own PSQA. Hence a new audit concept which assesses the institution's ability to detect errors with their routine PSQA is needed. METHODS: Purposefully introduced edits which simulated treatment delivery errors were embedded into radiation treatment plans of participating institutions. These were designed to produce clinically significant changes yet were mostly within treatment delivery specifications. Actual impact was centrally assessed for each plan. Institutions performed PSQA on each plan, without knowing which contained errors. RESULTS: Seventeen institutions using six radiation treatment planning systems and two delivery systems performed PSQA on twelve plans each. Seventeen erroneous plans (across seven institutions) passed PSQA despite causing >5% increase in spinal cord dose relative to the original plans. Six plans (from four institutions) passed despite a >10% increase. CONCLUSIONS: This novel audit concept evolves beyond testing an institution's ability to deliver a single test case, to increasing the number of errors caught by institutions themselves, thus increasing quality of radiation therapy and impacting every patient treated. Administered remotely this audit also provides advantages in cost, environmental impact, and logistics.

Predicted Risks of Cardiovascular Disease Following Chemotherapy and Radiotherapy in the UK NCRI RAPID Trial of Positron Emission Tomography-Directed Therapy for Early-Stage Hodgkin Lymphoma.

PURPOSE: The contemporary management of early-stage Hodgkin lymphoma (ES-HL) involves balancing the risk of late adverse effects of radiotherapy against the increased risk of relapse if radiotherapy is omitted. This study provides information on the risk of radiation-related cardiovascular disease to help personalize the delivery of radiotherapy in ES-HL. METHODS: We predicted 30-year absolute cardiovascular risk from chemotherapy and involved field radiotherapy in patients who were positron emission tomography (PET)-negative following three cycles of doxorubicin, bleomycin, vinblastine, and dacarbazine chemotherapy within a UK randomized trial of PET-directed therapy for ES-HL. Cardiac and carotid radiation doses and chemotherapy exposure were combined with established dose-response relationships and population-based mortality and incidence rates. RESULTS: Average mean heart dose was 4.0 Gy (range 0.1-24.0 Gy) and average bilateral common carotid artery dose was 21.5 Gy (range 0.6-38.1 Gy), based on individualized cardiovascular dosimetry for 144 PET-negative patients receiving involved field radiotherapy. The average predicted 30-year radiation-related absolute excess overall cardiovascular mortality was 0.56% (range 0.01%-6.79%; < 0.5% in 67% of patients and > 1% in 15%), whereas average predicted 30-year excess incidence was 6.24% (range 0.31%-31.09%; < 5% in 58% of patients and > 10% in 24%). For cardiac disease, the average predicted 30-year radiation-related absolute excess mortality was 0.42% (0.79% with mediastinal involvement and 0.05% without) and for stroke, it was 0.14%. CONCLUSION: Predicted excess cardiovascular risk is small for most patients, so radiotherapy may provide net benefit. However, for a minority of patients receiving high doses of radiation to cardiovascular structures, it may be preferable to consider advanced radiotherapy techniques to reduce doses or to omit radiotherapy and accept the increased relapse risk. Individual assessment of cardiovascular and other risks before treatment would allow personalized decision making about radiotherapy in ES-HL.

QSAR Study of Biologically Active Essential Oils against Beetles Infesting the Walnut in Catamarca, Argentina.

Essential oils from six species of aromatic plants collected in the Catamarca Province of Argentina were evaluated for their chemical composition and repellent and insecticidal activities against beetles of the genus Carpophilus (Coleoptera: Nitidulidae) and Oryzaephilus (Coleoptera: Silvanidae) that infest the local walnut production. Experimental data were analyzed using generalized estimating equations, with normal distribution and the identity link function. From the spectral information from the tested essential oils, we worked their molecular modeling as mixtures by developing mixture descriptors ( Dmix) that combined the molecular descriptor of each component in the mixture ( d i) and its relative concentration ( x i), i.e., Dmix = f( d i, x i). The application of chemoinformatic approaches determined that a combination of mixture descriptors related to molecular size, branchedness, charge distribution, and electronegativity were useful to explain the bioactivity profile against Carpophilus spp. and Oryzaephilus spp. The reported models were rigorously validated using stringent statistical parameters and essential oils reported with repellent activity against other beetle species from the Nitidulidae and Silvanidae families. This model confirmed each essential oil as a repellent with a comparable performance to the experimental reports.

MtMTP2-Facilitated Zinc Transport Into Intracellular Compartments Is Essential for Nodule Development in Medicago truncatula.

Zinc (Zn) is an essential nutrient for plants that is involved in almost every biological process. This includes symbiotic nitrogen fixation, a process carried out by endosymbiotic bacteria (rhizobia) living within differentiated plant cells of legume root nodules. Zn transport in nodules involves delivery from the root, via the vasculature, release into the apoplast and uptake into nodule cells. Once in the cytosol, Zn can be used directly by cytosolic proteins or delivered into organelles, including symbiosomes of infected cells, by Zn efflux transporters. Medicago truncatula MtMTP2 (Medtr4g064893) is a nodule-induced Zn-efflux protein that was localized to an intracellular compartment in root epidermal and endodermal cells, as well as in nodule cells. Although the MtMTP2 gene is expressed in roots, shoots, and nodules, mtp2 mutants exhibited growth defects only under symbiotic, nitrogen-fixing conditions. Loss of MtMTP2 function resulted in altered nodule development, defects in bacteroid differentiation, and severe reduction of nitrogenase activity. The results presented here support a role of MtMTP2 in intracellular compartmentation of Zn, which is required for effective symbiotic nitrogen fixation in M. truncatula.

Evolutionary History of the Hymenoptera.

Hymenoptera (sawflies, wasps, ants, and bees) are one of four mega-diverse insect orders, comprising more than 153,000 described and possibly up to one million undescribed extant species [1, 2]. As parasitoids, predators, and pollinators, Hymenoptera play a fundamental role in virtually all terrestrial ecosystems and are of substantial economic importance [1, 3]. To understand the diversification and key evolutionary transitions of Hymenoptera, most notably from phytophagy to parasitoidism and predation (and vice versa) and from solitary to eusocial life, we inferred the phylogeny and divergence times of all major lineages of Hymenoptera by analyzing 3,256 protein-coding genes in 173 insect species. Our analyses suggest that extant Hymenoptera started to diversify around 281 million years ago (mya). The primarily ectophytophagous sawflies are found to be monophyletic. The species-rich lineages of parasitoid wasps constitute a monophyletic group as well. The little-known, species-poor Trigonaloidea are identified as the sister group of the stinging wasps (Aculeata). Finally, we located the evolutionary root of bees within the apoid wasp family "Crabronidae." Our results reveal that the extant sawfly diversity is largely the result of a previously unrecognized major radiation of phytophagous Hymenoptera that did not lead to wood-dwelling and parasitoidism. They also confirm that all primarily parasitoid wasps are descendants of a single endophytic parasitoid ancestor that lived around 247 mya. Our findings provide the basis for a natural classification of Hymenoptera and allow for future comparative analyses of Hymenoptera, including their genomes, morphology, venoms, and parasitoid and eusocial life styles.