We have previously described that the NFκB pathway is upregulated during differentiation of glioblastoma stem-like cells (GSCs) which keeps differentiating GSCs in a proliferative astrocytic precursor state. However, extracellular signals and cellular mediators of this pathway are not clear yet. Here, we show that TLR4 is a key factor to promote NFκB activation in differentiating GSCs. TLR4 is upregulated during differentiation of GSCs and promotes transcriptional activation of NFκB as determined by luciferase-reporter assays and expression of NFκB target genes. Downregulation of TLR4 by shRNAs or blockade with anti-TLR4 specific antibodies drastically inhibited NFκB activity which promoted further differentiation and reduced proliferation of GSCs. We found that hyaluronic acid (HA), a main component of brain extracellular matrix, triggers the TLR4-NFκB pathway in differentiating GSCs. Moreover, HA is synthesized and released by GSCs undergoing differentiation and leads to transcriptional activation of NFκB, which is inhibited following downregulation of TLR4 or blockade of HA synthesis. Thus, we have demonstrated that during the process of differentiation, GSCs upregulate TLR4 and release the TLR4 ligand HA, which activates the TLR4-NFκB signaling pathway. This strategy may efficiently be used by differentiating GSCs to maintain their proliferative potential and consequently their tumorigenic capacity. A number of antitumor strategies induce DNA damage, or inhibit cell division and angiogenesis, mainly by small molecular inhibitors or blocking antibodies. Activation of DNA damaging mechanisms by genotoxic agents may induce apoptotic or senescent cell death2. Differentiation-inducing agents offer an alternative antitumor strategy and some compounds are able to increase the efficacy of chemotherapy or radiotherapy4. Based on this, strategies that promote differentiation and senescence provide therapeutic opportunities that need to be further explored.Glioblastoma is a highly aggressive tumor with poor survival rates and treatment remains a challenge. Work in different types of cancer, including glioblastoma, suggest that cancer stem-like cells are resistant against radiotherapy and chemotherapy, which facilitates tumor recurrence5. It has also been shown that inhibition of NFκB facilitates stemness6, and triggers proliferation of neural stem cells8. Proinflammatory signals like cytokines and pathogen-associated molecular patterns (PAMP) trigger an intracellular mechanism that leads to the activation of NFκB9. Several of these signaling pathways are initiated by activating cell surface receptors, including tumor necrosis factor receptor and Toll-like receptor (TLR) superfamilies. At present, 11 mammalian TLRs have been described. TLR proteins recognize PAMPS that include bacterial lipopolysaccharides and peptidoglycans or viral RNAs among others. Upon binding of the ligand, TLR proteins recruit a signaling adaptor protein, mainly MyD88, that activates a kinase cascade which ultimately promotes activation of the NFκB protein complex that is translocated to the nucleus to induce the expression of target genes. TLR4 is one of the most studied members of the TLR family and it has been involved in inflammation and resistance to virus, as well as in tumor microenvironment. TLRs, including TLR4, have been shown to be overexpressed in breast cancer10 and tumor cells from a wide variety of tissues11 suggesting that TLR activation may be an important event in tumor cell immune evasion. Activation of TLR4 in tumor cells promotes the synthesis of NFκB target genes, including IL-6 and IL1β, which results in resistance of tumor cells against cytotoxic lymphocytes. However, the role of the TLR4-NFκB signaling pathway during differentiation of cancer stem-like cells has been poorly studied.There are many signaling pathways that promote oncogenic transformation or maintain the aggressiveness of tumor cells. Some of these pathways induce activation of NFκB, a transcription factor involved in many cellular processes including cell survival, proliferation and migrationHere we show that TLR4 is upregulated during differentiation of GSCs, which triggers the NFκB transcriptional pathway, avoiding terminal differentiation and maintaining proliferation. This cell behaviour is reversed following downregulation or inactivation of TLR4. We also show that TLR4 is activated by hyaluronic acid (HA) that is synthesized and secreted by differentiating GSCs.Blocking anti-TLR4 antibodies and control IgG2a  were mostly used at 0.5 μg/ml in cell cultures. HA fragments with a size distribution of 15–40 kDa  were added at a concentration of 100 μg/ml when indicated. HA synthesis inhibitor 4-Methylumbelliferone (4-ME)  was added to cell cultures at 2 mM. HA levels in cell culture supernatants were determined by an ELISA kit from Echelon Biosciences .12. Briefly, tumor cells were obtained following digestion of tissue samples and cultured in serum-free DMEM/F12 medium  with growth factors containing human recombinant EGF . To induce differentiation, neurospheres were disaggregated into single cells that were cultured as a monolayer in the same medium but containing 10% FCS for the indicated time intervals as previously described13. For cell proliferation assays, neurospheres were disaggegated into single cell suspensions before cell growth was analysed at the indicated time intervals by using Alamar blue reagent .GSCs were derived from patients with primary glioblastoma with wild-type IDH1 and EGFR amplification. All the results shown in figures have been obtained from a single cell culture, but in all cases similar data were replicated in one or two more GSC cultures. Neurosphere cultures were established as previously describedBriefly, differentiating GSCs were grown on 10 × 10 mm coverslips. Cells were then fixed in 3.7% formaldehyde and permeabilized with 0.5% Triton X-100. Then, cells were incubated with rabbit anti-GFAP  and anti-Ki67  or mouse anti-phospho histone H2AX  antibodies. Texas red-conjugated or FITC-conjugated goat anti–rabbit or anti-mouse secondary antibodies  were used for detection. For fluorescence quantification, markers were analyzed by using the AxioVision software (Carl Zeiss Inc.). Nuclear area was quantified using ImageJ software  on at least 100 DAPI-positive nuclei.12. Differential expression of all TLR family members included in the array was analyzed using the fold-change value. Gene expression was assessed by using primers for TLR4 (5′AGTTTCCTGCAATGGATCAAGG3) and (5′CTGCTTATCTGAAGGTGTTGCAC3), Map2 (5′TTCGTTGTGTCGTGTTCTCA3′) and (5′AACCGAGGAAGCATTGATTG3′), TNFα (5′CGGGACGTGGAGCTGGCCGAGGAG3′) and (5CACCAGCTGGTTATCTCTCAGCTC3′), IL-10 (5′CCGAGATGCCTTCAGCAGAG3′) and (5′CACATGCGCCTTGATGTCTG3′), IL-6 (5′AGTGAGGAACAAGCCAGAGC3′) and (5′GAGATGAGTTGTCATGTCCTGC3′), and β-Actin (5′GCGGGAAATCGTGCGTGACATT3′) and (5′GATGGAGTTGAAGGTAGTTTCGTG3′). Quantitative PCR was performed as previously described14.Gene expression analysis comparing neurosphere-forming GSCs and GSCs differentiated in the presence of 10% FCS for 4 days, was performed with the Human Genome U133 Plus 2.0 array  as describedDetection of the levels of TLR protein was achieved by using a TLR cell surface screening set  that included specific antibodies against TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10. For each individual analysis, an isotype antibody was used for non-specific staining. PE-labeled secondary antibodies were then used and labelled cells were analysed by flow cytometry  using the ModFit software . TLR expression levels are shown as mean fluorescence intensity subtracting the corresponding isotype control.12 and 50 ng of pRSV-β-gal by nucleofection . Cells were also transfected with 1 μg of pBVIx-Luc, a plasmid containing NFκB consensus sequences linked to the luciferase reporter gene. Luciferase activity was analysed by a dual-light reporter gene assay (Applied Biosystems). Results were normalized by cotransfection with pRSV-β-gal.GSCs were co-transfected with 1 μg of pGL2-MAP2 promoter construct4 cells per well in a 96-well plate. Five non-overlapping TLR4-specific or control scrambled shRNA-containing lentiviral particles were used  as indicated by the manufacturer. As a control of transduction efficiency, GSCs were infected with lentivirus containing shRNA to EGFP.GSCs were dissociated from neurospheres and cultured at a density of 2 × 10Statistics were analyzed with the SPSS package (version 13.0). The Student t test was used to compare continuous variables as means ± SD, between two groups. The significance level was set at p < 0.05.12. Consistently, GBM is characterized by poorly differentiated cancer cells with high mitotic activity15. The terms differentiating cells or cells undergoing differentiation, refer to a population of proliferative astrocyte precursors without stellate morphologies and with low levels of stem cell markers and increased expression of GFAP astrocytic marker. It is also well known that TLR signaling pathways promote activation of NFκB16. Our first goal was to study the expression of TLR family members in differentiating GSCs. Based on our microarray gene expression analyses of GSCs before and after differentiation for 4 days12, TLR4 was the only TLR member that increased its levels in all five cell lines tested  avoiding terminal differentiation and promoting proliferationted Fig. . Flow cyted Fig. . Downregted Fig. . Then, Tted Fig. . A well lls Fig. . In ordells Fig. . Downreglls Fig.  and a sills Fig. . NFκB palls Fig. . This re12. In order to assess whether downregulation of TLR4 modified the differentiation pattern of GSCs, we first analyzed the expression of GFAP, an astrocytic differentiation marker, and found that low levels of TLR4 correlated with increased expression of GFAP (about 3-fold higher intensity with shRNA-1 relative to control) as determined by immunofluorescence  and nuclei showed an accumulation of phospho-H2AX in approximately 24% of cells  and neuronal (22–45%) phenotypesnce Fig. . Transfence Fig. . Additionce Fig. . In linence Fig. . Consistnce Fig.  as deternce Fig. . Two fealls Fig. . Similarlls Fig. .Figure 321, and has been shown to signal through TLR422. We showed that exogenous HA increases the NFκB transcriptional activity in differentiating GSCs and this activation was lost following downregulation of TLR4 by shRNAs  (data not shown). However, if we block the production of HA with 4-Methylumbelliferone (4-MU), a specific HA synthesis inhibitor used in many in vitro studies27, the secretion of HA by differentiating cells was reduced to levels similar to those of GSCs  is a main component of the extracellular matrix in the brainNAs Fig. . We haveted Fig. . Moreoveted Fig. . We haveSCs Fig. . ConsistSCs Fig.  and the SCs Fig.  of diffeSCs Fig. . Thus, H28. We have previously demonstrated that canonical NFκB signaling, that is poorly activated in GSCs, is upregulated during differentiation of GSCs which allows cells to proliferate and avoid terminal differentiation and senescence12. This is consistent with previous work showing that canonical NFκB pathway has no or very little effect on GSCs29. In addition, activation of canonical NFκB has also been associated with mesenchymal transdifferentiation and radioresistance of a subset of GSCs31. However, the stimuli that trigger this activation in differentiating GSCs are not well defined. We showed that TLR4 was the only TLR member consistently upregulated in all differentiating GSC cultures established from surgical specimens of GBM patients. In line with this, it has been recently described that cancer stem cells isolated from patient-derived GBM xenografts showed a gradual increase in the expression of TLR4 under differentiation conditions13. TLR4 signaling results in activation of the NFκB pathway in astrocytes, microglia, neurons and neural progenitors32. Consistently, we demonstrated that TLR4 upregulation in differentiating GSCs was associated with increased NFκB transcriptional activity that was blocked by downregulating the levels of TLR4 or treatment with neutralizing TLR4 antibodies. Moreover, activation of the TLR4-NFκB axis in differentiating GSCs promoted proliferation of these cells and avoided terminal differentiation and senescence, a phenotype that characterizes GBM tumors. Experimental support of this phenotype comes from GSC xenograft models, that showed a clear absence of fully differentiated morphology indicating that tumor cell populations comprise of progenitor cells that have not completed astrocytic differentiation24. Previous data also showed that astrocytic precursors have a relatively high proliferation rate25. Consistent with our data, activation of the NFκB pathway is low in embryonic stem cells, but increases after differentiation33. However, it is not known whether this activation relies on a TLR4-mediated pathway. The low activation of canonical NFκB signaling in GSCs indicates that NFκB may be dispensable for survival and proliferation of stem cells, and this is consistent with a predominant accumulation of NFκB proteins in the cytoplasm of cells within neurospheres from embryonic mouse brain7. Recent data have shown that low TLR4 expression in GBM cancer stem cells allows these cells to survive by disregarding inflammatory signals13. Differentiated tumor cells would not have this adaptive advantage. However, on the other hand, a sustained activation of TLR4-NFκB will provide proliferation signals and some of the newly generated cells may develop biological mechanisms that enable them to migrate to more favorable microenvironments or survive to the immune attack. To this end, tumor heterogeneity is one of the most important hallmarks of GBM. Different cell subclones have been established from a GBM patient and all of them behave differently in terms of morphology, proliferation, response to treatment and aggressiveness in animal models34. NFκB has been shown to induce and also to inhibit differentiation indicating a broad spectrum of activities depending on the cell type38. We showed that blockade of TLR4-NFκB signaling by using genetic strategies or blocking antibodies accelerates maturation of differentiating GSCs. Previous data indicate a role of NFκB in terminal differentiation. For instance, activation of NFκB pathway induces cell division and inhibits differentiation of mammary epithelial cells39. Moreover, in vivo inhibition of NFκB signaling by using a small molecule induced sensescence of tumor cells in mice bearing GSC-derived tumors12.GSCs have been identified as key cells for tumor initiation and recurrence, providing therapeutic opportunities40, and there are several examples showing that HA signals through TLR441. We found that HA promotes activation of NFκB through TLR4 in differentiating GSCs when added to cell cultures. However, we observed an increase in NFκB activity in GSCs maintained under differentiation conditions without exogenous HA. Interestingly, we demonstrated that GSCs produce and secrete their own HA and this production is highly increased in differentiating cells. Blockade of HA synthesis abrogated the TLR4-NFκB pathway indicating that GSCs develop their own mechanism to assure activation of NFκB during the differentiation process. This is consistent with previous work showing that a human GBM cell line synthesized and secreted HA into the medium42. These authors also found that HA synthesis by glioma cells occurred during the proliferative phase, which is in line with our model of differentiating GSCs where upregulation of TLR4 and secretion of HA by intermediate precursor cells activate the NFκB transcriptional pathway. NFκB promotes proliferation by inducing the expression of growth factors such as IL1β or IL643 and we showed that differentiating GSCs increased the levels of IL6 which were abrogated in the presence of a HA synthesis inhibitor. Thus, immature GBM cells may use this HA-TLR4-NFκB pathway as a mechanism to allow proliferation and avoid terminal differentiation and senescence. Although there are a number of studies demonstrating that HA signals via TLR444 and even suggesting HA-TLR4 interactions22, another study claimed that HA was not a ligand but a regulator of TLRs45. Two relevant issues may help explain these conflicting results. First, those studies that suggest a ligand-receptor scenario used HA fragments with molecular size above 100 kDa, whereas the study arguing that HA is a TLR4 regulator used HA fragments below 3 kDa and this is an important issue as HA biological activities differ widely depending on the fragment sizes46. Additionally, all these works used different cell models, including glomerular mesangial cells, macrophages, dendritic cells and endothelial cells which may have different responses to HA. Further biochemical studies will be needed to undoubtedly identify HA as a cognate TLR4 ligand. It would also be interesting to study the relevance of the HA-TLR4 pathway in other in vitro or in vivo models that provide a more physiological context. In this regard, 3D cultures such as the spheroid model have been used for evaluating drug efficay or response to antiangiogenic conditions49. However, the cellular heterogeneity of GBM may need more complex in vitro systems or in vivo xenograft models to more closely mimic the native tumor microenvironment.At this point a key issue is how do GSCs undergoing differentiation activate the TLR4-NFκB pathway. It has been shown that hyaluronic acid (HA) is among the key organizers of the brain extracellular matrixOverall, we have demonstrated that TLR4 is upregulated during the differentiation process of GSCs and that TLR4 promotes activation of the canonical NFκB pathway in these cells. TLR4-NFκB signaling is mainly orchestrated by HA that is synthesized and secreted by differentiating GSCs. Our results provide new findings for therapeutic opportunities aimed to promote terminal differentiation of GSCs through inhibition of the NFκB signaling pathway.


In the Carnian (Late Triassic), evidence from sedimentology and fossil pollen points to a significant change in climate, resulting in biotic turnover, during a time termed the ‘Carnian Pluvial Episode’ (CPE). Evidence from the marine realm suggests a causal relationship between the CPE, a global ‘wet’ period, and the injection of light carbon into the atmosphere. Here we provide the first evidence from a terrestrial stratigraphic succession of at least five significant negative C-isotope excursions (CIE)’s through the CPE recorded in both bulk organic carbon and compound specific plant leaf waxes. Furthermore, construction of a floating astronomical timescale for 1.09 Ma of the Late Triassic, based on the recognition of 405 ka eccentricity cycles in elemental abundance and gamma ray (GR) data, allows for the estimation of a duration for the isotope excursion(s). Source mixing calculations reveal that the observed substantial shift(s) in δ The CPE is characterised by elevated extinction rates in the marine realm, and a contemporaneous increase in terrestrial species diversity with a temporary switch to more hygrophytic flora8. Subsequently, the late Carnian marks the dawn of calcareous nanoplankton and scleractinian reef builders and the rise of the early dinosaurs9.The Late Triassic period represents a time of extreme aridity and relative environmental and climatic stability, interrupted only by a brief, but substantial switch to more humid conditions, followed by ecological crises in the middle Carnian, during the CPE15. Contemporaneously, in the Germanic Basin, sabkha sediments are replaced by predominantly fluvial deposits of the Stuttgart Formation (Schilfsandstein)16. Furthermore, marine records indicate the establishment of oxygen depleted conditions in marginal basins, with an increase in siliciclastics, and a temporary shutdown of carbonate systems across the Tethyan realm  and a compound specific (4‰) negative CIE at the onset of the CPE suggests the injection of 13C-depleted CO2 into the Earth system3. Nevertheless, no carbon isotopic evidence for the CPE from a terrestrial stratigraphic succession exists. Moreover, the timing and duration of the CPE is still unclear thereby hampering the establishment of a causal relationship between the mechanism, the CIE, and the associated environmental change. Here we present for the first time continental records of: i) bulk sediment δ13C, and ii) compound specific δ13C of plant leaf waxes  through the Late Triassic (Carnian) of the Wiscombe Park borehole , to assess the CPE within the terrestrial realm. Additionally, cyclostratigraphic investigation using elemental abundance and GR data is used to establish a floating time-scale for the succession, thus constraining the duration of the CIEs.Despite the global significance of the CPE as a major environmental perturbation, the trigger of the environmental change and subsequent biotic turnover is still disputed. Evidence from the marine realm of the existence of both a bulk  ranges from −30.1‰ to −25.1‰  but during the DMF fluctuates highly  is followed by a further four transitory negative CIEs within the DMF, with a return to heavier values of c. −22.3‰ from 50 m to the core top  to confirm the trends of the δ13CTOC record. Leaf waxes, showing a clear odd-over-even predominance, are produced by higher plants and considered stable once buried in sediments18. Hence, variations in their isotopic composition unambiguously record past climate variability19 (see supplementary information). δ13Cwax values range from −25.7‰ to −34.3‰ and are depleted by c. 5‰ with respect to bulk δ13CTOC  through the SMF during the Carnian. This trend has been linked to the re-emergence of coal swamps and increased carbon burial rates20.The total organic carbon (TOC) contents varies between 0.04 and 7.7% Fig. , and the.1‰ Fig. . The δ13hly Fig. . δ13CTOCtop Fig. . Since tTOC Fig. . The IIEore Fig. . Althoug10 reveals statistically significant regular cycles throughout the lacustrine DMF for both WP borehole 1 & 2  Carnian climate oscillations are related to long and short eccentricity cycles as well as precessional variability26. Cyclicity is less clear within the XRF data of the SMF  elemental abundance and GR data& 2 Fig. . Within cle Fig. . This seSMF Fig.  and in t3, we identify a further four appreciable negative CIE’s within the CPE  required from each source to explain the observed negative CIE: (9540 Gt C + n Gt C) × (δ13Catmosphere early Julian II) = 9540 Gt C × (δ13Catmosphere late Julian I) + n Gt C × (δ13Cemission). Our calculations indicate that even a complete collapse in primary productivity  would only account for around one quarter of the carbon required (c. 3500 Gt C) to produce the observed δ13C isotope shift. It is plausible that the CPE was associated with a crisis in marine bioproductivity, further supported by the existence of a widespread demise in reef ecosystems17; however, insufficient evidence for such an extensive collapse exists. The CPE excursion would require an additional 14,000 Gt C of magmatically derived CO2 (using δ13C = c. −8‰29). However pools of lighter carbon in the mantle, as well as peridotic and eclogitic diamonds, have been measured with δ13C values as low as −38–−22‰31. If the δ13C value is set to −20‰, then only c. 3500 Gt C is required to produce our observed isotope excursion. With estimates of 5000 Gt C released during the eruption of the Wrangellia igneous province alone3, a pure volcanic scenario is possible. Alternatively, if volatilized organic carbon was the primary source of the light-C, then c. 2700 Gt C would have been required to produce the observed excursion (δ13C = c. −25‰32), the equivalent of all present-day terrestrial carbon stocks33. Nevertheless, there is little evidence for substantial contact metamorphism of carbon-rich sediments within petroleum-bearing basins during the CPE. Conversely, just a small contribution (c. 1000 Gt C) from methane clathrates (δ13C = c. −60‰34) would have been required to produce the observed excursion. Estimates indicate that marine sediments at present contain about 10,000 Gt of methane carbon, thus a release of 1000 Gt C is plausible35. The stability of methane hydrates is temperature dependent, with warming leading to destabilisation. Indeed, we observe evidence for climatic warming during the CPE at sites from the eastern and northwestern Tethys36. Such methane release would quickly be sequestered by both terrestrial and marine components of the global carbon cycle, and would contribute to the widespread deposition of the black shales evidenced during the CPE5.Unlike the marine succession from the Dolomites where one CIE is identified13C, a scenario comparable to that of the End Triassic Extinction (ETE)37. We envisage episodic, punctuated release of methane from clathrates during progressive warming caused by the Wrangellia eruptions.Volcanic emissions from the Wrangellia igneous province and the dissociation of methane clathrates could independently account for the observed multiple negative CIE’s within CPE. Nevertheless, a causal relationship likely exists between the Wrangellia eruption and the subsequent destabilization of methane clathrates. It seems that a combination of both volcanic emissions and the dissociation of methane clathrates would be the most likely explanation for the substantial observed shift(s) in δ39), for example: (i) a substantial IIE , (ii) a similar duration of IIE , (iii) a negative positive couplet , and (iv) a return to oscillating but generally depleted values throughout the excursion. The end Permian excursion shows again a sharp, short initial excursion, however the swing back toward heavier values is much smaller than at the CPE and ETE, and values remain some 0–1.5‰ lighter  sediments from Devon (UK) provide the first evidence from non-marine strata of the negative CIE observed, coinciding with the CPE extinction event. Furthermore, in contrast to marine successions, we identify not one, but five, significant isotope excursions. By utilizing the persistent presence of strong 405 ka eccentricity cycles through the 20.4 m record of the DMF we have constructed an astronomical timescale for 1.09 Ma years of the Late Triassic, allowing us to estimate a likely duration for the C-isotope excursions associated with the CPE. Through source mixing analysis we calculate that a combination of volcanic emissions and subsequent methane release were the likely cause for the observed shift in δ13C and the associated extinction event.Compound specific and total organic carbon δ13C was analysed using an Agilent 6890 gas chromatographer coupled with a Thermo Finnigan DeltaPLUSXL isotope ratio mass spectrometer (GC-C-IRMS). Ratios were calibrated daily based on the reference standard Schimmelmann B. For δ13CTOC analyses, samples from WP borehole 1 were decalcified, neutralized and subsequently dried. Samples were measured for % TOC using a Fisons NA1500 NCS and coupled with a Thermo Delta plus IR-MS for stable C-isotope analyses. Ratios were normalised using the laboratory standard GQ to the V-PDB standard. Major element abundances were attained using a hand-held Niton XRF analyser at the BGS. MTM, F-test, bandpass and ASM were performed on the XRF data from WP borehole 1 and GR data from WP borehole 2 using the R program Astrochron43. The Continuous Wavelet Transform was achieved using a Morlet wavelet and run on a MATLAB platform. Further technical information for all methods is provided in the supplementary information.Sediments from WP borehole 1 were extracted with dichloromethane (DCM):MeOH  using soxhlet extraction. The total extract was separated into apolar and polar fractions over an alumina oxide column, eluting with hexane:DCM  and DCM:MeOH , respectively. Compound δSupplementary information


Cyp19a1 expression and aromatase protein levels only in female neuronal cultures. The effect of E2 on aromatase expression was not imitated by oestrogen receptor (ER) α agonist PPT or the GPER agonist G1, but it was fully reproduced by DPN, a specific ligand of ERβ. By contrast, the effect of DHT on aromatase expression was not blocked by the anti-androgen flutamide, but completely abrogated by the ERβ antagonist PHTPP. Experiments using the four core genotype model showed a sex chromosome effect in ERβ expression (XY > XX) and regulation by E2 or DHT (only XX respond) in amygdala neurons. In conclusion, sex chromosome complement governs the hormonal regulation of aromatase expression through activation of ERβ in developing mouse brain.During development sex differences in aromatase expression in limbic regions of mouse brain depend on sex chromosome factors. Genes on the sex chromosomes may affect the hormonal regulation of aromatase expression and this study was undertaken to explore that possibility. Male E15 anterior amygdala neuronal cultures expressed higher levels of aromatase (mRNA and protein) than female cultures. Furthermore, treatment with oestradiol (E2) or dihydrotestosterone (DHT) increased  The noteworthy evidence that testosterone (T) influences sex-specific behaviours through organisation of the developing brain during a critical period early in development1, complemented with later evidence that oestradiol (E2) is an important metabolite in the brain2 has established the basis for the latter studies. More recently sex chromosome genes have been recognized as the primary factors causing sexual differentiation  not only as direct effectors of some sex specific traits but also acting in parallel with sex steroids. Aromatase, one of the last enzymes of neurosteroidogenesis, is responsible for the conversion of T into E2. The expression of aromatase is limited to sexually dimorphic regions of the brain6 and presents sex differences during the sensitive period of sexual differentiation4.The field of sexual differentiation has been receiving increasing contributions since the pioneering work of Phoenix 7 (E16). These and previous findings of higher activity and expression of aromatase in hypothalamic regions of male rat and mouse brain at E1611 are not determined by the prenatal increase in T production by the foetal testis, which in rats occurs at E18.5–19.513 and in mice at E17–1815. Importantly, the use of the “Four Core Genotypes” (FCG) mouse model in our previous study7 allowed us to establish that sex chromosomes influence aromatase independently of gonadal status. This mouse model combines a spontaneous deletion of the sex determining region Y (Sry gene) from the Y chromosome (Y−) with the re-insertion of a functional Sry transgene onto an autosome17. The XY−Sry mice have testes and are fully fertile. The Sry transgene and the Y− chromosome separate independently during meiosis, thus, four genotypes are present in the offspring: XX females, XY− females, XXSry and XY−Sry male mice.In concordance with previous results we recently demonstrated a sex chromosome-induced difference in aromatase expression in the anterior amygdaloid area and the stria terminalis of the mouse brain as early as embryonic day 1622. While some evidence indicates that androgens and oestrogens synergize to induce aromatase mRNA in the quail brain23, other evidence indicates that dihydrotestosterone (DHT) seems to be more effective than T in the induction of aromatase21. Importantly, in well-known sexually dimorphic brain regions such as the preoptic area and hypothalamus, testosterone and DHT induce aromatase expression24. Our previous study using amygdala neuronal cultures indicates that E2 and DHT regulate aromatase expression through a mechanism controlled by sex chromosome complement. E2 and DHT increase aromatase mRNA levels in cultures of anterior amygdala neurons coming from XX embryos7. Since oestrogen (ER) and androgen (AR) receptors are critical for steroid hormone effects during brain development and their expression mostly corresponds to brain regions which express aromatase25, brain masculinization requires ERs in some brain regions and AR function in others. In addition, although ERα has been implicated in the masculinizing actions of oestradiol in hypothalamic and preoptic areas26 less is known about ERβ involvement during development.The regulation of aromatase expression by gonadal steroids has been well documentedIn the present study we evaluated whether sex chromosome factors determine the mechanism by which sex hormones regulate aromatase expression in the amygdala. Our findings indicate that differential expression of ERβ is regulated by sex chromosomes, and underlies the hormonal regulation of neuronal aromatase expression in the amygdala. Given that this study was carried out before the critical period of increased gonadal secretion, these findings imply that genetically controlled mechanisms precede gonadal influences during the genesis of differences between the sexes in brain structure.in vivo7, male neuronal cultures of E15 CD1 embryos expressed higher levels of Cyp19a1 mRNA . Similar results were obtained for aromatase protein levels. Two-way ANOVA revealed a significant sex-by-treatment interaction . One-way ANOVA showed a significant main effect of treatment . The treatment of female cultures with the ERα agonist PPT or the GPER agonist G1 did not significantly affect Cyp19a1 levels (p = 0.77 and p = 0.20 respectively). We further examined the effect of E2 in combination with the selective ERβ antagonist PHTPP. One-way ANOVA demonstrated a main effect of treatment , and PHTPP blocked the effect of E2 on Cyp19a1 levels .In order to identify the ER subtype involved in the regulation of Cyp19a1 mRNA levels in amygdala neuronal cultures originating from E15 female mice7. In view of this finding, the same experimental procedure was applied to amygdala neuronal cultures in order to determine the mechanism involved in this regulation.Our previous results showed the expression of 5α-reductase enzymes I and II in punches of the amygdala from E16 male and female mouse embryos. Therefore, the amygdala has the enzymatic machinery to synthetize DHT from T. In addition, we have shown that the treatment with DHT resulted in an increase in Cyp19a1 levels were increased in female cultures to male control levels . Similar results were obtained for aromatase protein levels . Cyp19a1 levels in female cultures treated with DHT + flutamide remained high as in DHT alone (LSD test: p = 0.289).To determine whether DHT regulates Cyp19a1 mRNA levels  while in XX neurons Esr2 expression was significantly increased (p = 0.027) with an overall effect of abolishment of genotype-induced sex differences between XX and XY neurons (p = 0.69). Interestingly, FCG neuronal cultures stimulated with DHT demonstrated a similar regulation for ERβ mRNA. DHT treatment resulted in a genotype-by-treatment interaction in the ANOVA  but not in XY cultures (p = 0.38). Thus, both hormonal treatments counteracted the sex chromosome effect making XX cultures treated with E2 or DHT equivalent to XY control cultures.The results above demonstrate that E2 and DHT regulated aromatase expression only in female neurons through a mechanism involving ERβ activation. We therefore asked whether the sex chromosome complement that determines the capacity for aromatase regulation is associated to the regulation of ERβ in XX neurons. To deal with this question, ERβ expression was assessed in amygdala neuronal cultures from E15 embryos belonging to FCG mice. As shown in Fig. 1,31 = 18.31; p = 0.0002), sex chromosome complement , and interaction of both factors  on Esr1 expression. XX female cultures showed higher expression levels of Esr1 and E2 stimulation did not significantly change Esr1 mRNA . They showed that gonadal/hormonal upregulation of aromatase expression occurred in both sexes, whereas in our study only females respond to E2 or DHT with augmented levels of aromatase suggesting differential hormonal sensitivity of amygdala between prenatal period and adulthood. Although our findings show an all or none sex dimorphic response to E2 and DHT, it is important to mention that we have assessed only a single time point (2 h post-treatment) and a single dose of the hormones. Subtler differences in the sensitivity of male and female amygdala neurons to E2 and DHT may be revealed by assessing additional doses and time points.Regulation of brain aromatase by gonadal hormones in rodents has been demonstrated previously29 that could be expressed differently in both sexes at E15–16. Previously we have shown a higher AR mRNA expression in the anterior amygdala of male mice, compared to females, at E167. Remarkably, the expression of ERα mRNA in the same region was higher in female than in male mice7. In developing rats ERα mRNA levels are present in preoptic and amygdala regions since E1830. In addition, it was described that ERα and ERβ are expressed in posterodorsal part of the medial amygdaloid nucleus during early postnatal development31. In order to unravel the participation of classical and non-classical steroid receptor-dependent mechanisms we assessed several compounds that act as agonists or antagonists of AR and ERs. DPN, an ERβ agonist, imitated the effect of E2 on aromatase expression. Moreover, the effects of E2 were blocked by co-administration of the ERβ antagonist, PHTPP. By contrast, PPT and G1, selective agonists of ERα and GPER, respectively, were not able to imitate the effect of E2 on aromatase expression. Furthermore, the effect of DHT on aromatase was not antagonized by flutamide, a selective AR antagonist; but was completely blocked by PHTPP, the selective antagonist of nuclear ERβ. All these findings suggest that the hormones E2 and DHT are regulating aromatase expression by a classical nuclear ERβ mediated transcription.There are several possible explanations for the different response of male and female neurons to exogenous hormones. One important consideration is that amygdala regions are rich in nuclear AR and ERs32 which would lead to a sexually differential regulation of the enzyme itself. Although it was not possible to assess ERβ protein levels, due to the lack of adequate antibodies33, our study strongly supports that ERβ participates in the regulation of aromatase in amygdala at E15.Based on these results, we directly evaluated ERβ mRNA in our sexually segregated amygdala culture system. Surprisingly, we found a sex difference in ERβ mRNA expression in favour of males. This finding appears to be counterintuitive to results demonstrating that only female neurons were responsive to hormone treatments. One possible explanation for this apparent contradiction is that ERβ may be under different hormonal regulation in males and females. To address this issue we evaluated ERβ mRNA in amygdala cultures of FCG mouse model treated with E2 or DHT. Neurons with the XY sex chromosomes showed higher ERβ expression levels than neurons with XX sex chromosomes, irrespective of gonadal type. Hormonal treatments increased ERβ only in XX, resulting in the abolishment of sex differences in ERβ expression by a mechanism that also depends on genetic factors. A complementary and/or alternative explanation is that, in males, E2 may be degraded into catechol-oestrogens via hydroxylation by aromataseet al.35 indicate that DHT, in a fashion similar to T, may be converted to products with oestrogen-like activity by other enzymes than aromatase. One of these products is 3β-diol which preferentially binds to ERβ, and not to AR, often through a canonical ERE in the promoter of a given target gene36. Here we have found that the increase in aromatase expression by DHT could be blocked by co-administration of the ERβ antagonist, PHTPP, but not by the AR antagonist, flutamide. Furthermore, 3β-diol treatment to amygdala female cultures increased aromatase levels. Together, these results provide correlative evidence that 3β-diol mediates the effects of DHT on aromatase expression by binding ERβ. These findings may also explain the contradictory results found by other groups in amygdala region in which DHT seems to be more effective than T in the induction of aromatase21. Harada et al.23 have demonstrated that androgens and oestrogens synergize to induce aromatase mRNA in the quail brain. Also in the preoptic area and hypothalamus of mouse brain T and DHT induce brain aromatase expression24. This endocrine pathway has been shown to exist in numerous species and tissues37, but its functional significance was first suggested for prostate38, where it has been proposed that 3β-diol is the predominant endogenous oestrogen. In teleost fish, the regulation of the cyp19a1b gene by DHT also involves its conversion into 3β-diol39. The fact that gonadal/hormonal up regulation of aromatase depends on ERβ parsimoniously explains why E2 and DHT produce the same effect on aromatase expression.DHT, a 5α-reduced form of testosterone, has been classically considered a prototypical AR agonist with no ability to be aromatized to oestrogen-like metabolites. However, several reports reviewed by Handa 41 suggesting that its regulation is through classical steroid receptor-dependent mechanisms. Consistent with this, results from Pak et al.36 show that 3β-diol significantly increased ERE-mediated promoter activity to levels greater than that of E2, whereas it had no effect at AP-1 site. Yilmaz et al.42 using a hypothalamic neuronal cell line demonstrated that E2 positively regulates the brain-specific promoter of aromatase gene via a mechanism involving ERα. Our present results in amygdala neurons of developing mouse brain indicate that androgens and oestrogens increase aromatase expression through a common mechanism that involves the action of 3β-diol and the activation of ERβ, highlighting the complex region-specific mechanism that regulates aromatase expression in the brain.The aromatase gene contains androgen and oestrogen response elements7. In this study, we have found that hormonal regulation of aromatase in amygdala neurons depends on ERβ signalling. It could be hypothesized that the differential aromatase expression produces an imbalance in E2:T ratio between XY/XX brain amygdala during development  of sex chromosomes in males and females. The FCG mouse model allowed us to preclude the effect of Sry gene on the expression of ERβ since we found a significant difference between XX male (Sry+) neurons compared with XY male (Sry+) neurons. However, other Y genes have an inherently male function because they act on germ cells in a cell-autonomous fashion and are required for spermatogenesis; a male-specific function43. The latter could be interpreted as a possible explanation in the significant differences found between XY female and XX female neurons. It could also be possible that differences in the number of X chromosomes cause the sex differences in aromatase and ERβ expression. As it was recently reviewed by Arnold3, sex differences in gene expression also arise due to the female specific expression of Xist, a higher expression of X escapee genes in XX than in XY cells, the sexual inequality of parental imprints on X chromosome genes or due to female-specific effects of the inactive X chromosome. Some genes present in X or Y chromosomes encode for transcriptional regulatory proteins which are able to produce changes in chromatin remodelling and regulate autosomal gene expression44. In line with this, Nugent et al.45, have reported that aromatase gene Cyp19a1 is subjected to a sex-related differential DNA methylation in rat preoptic area during the critical period of sexual differentiation. Also the DNA methylation in CpG islands present in the promoter region of ERβ rat gene was reported to be differentially regulated across the life span in a manner specific to the brain region, age, sex, and neonatal hormone exposure of the animal46.The sex chromosome-induced sex differences found for ERβ expression in amygdala neurons could be interpreted as an additional mechanism by which sex chromosomes regulate aromatase expression ensuring adequate levels of ERβ in XX neurons for aromatase regulation during development. The fact that ERα and AR expressions were not subjected to hormonal regulation supports this interpretation. The regulation of both, 7 could be related to a neuroprotective mechanism evolved to ensure adequate levels of aromatase in XY brain during development. As it was previously stated, the autism spectrum disorder, a sex-biased neurodevelopmental disorder which occurs two to four times more often in boys and men than in females  has been related to environmental factors, especially the intrauterine developmental environment. Results from Xu et al.48 indicate that the prenatal hyperandrogenic environment produced by letrozol treatment induces autistic-like behaviour in rat offspring. Future studies could explore the upstream pathways involved in this mechanism.Sex specific regulation of aromatase expression reported here and in our previous studyIn summary, we have demonstrated that hormonal and genetic factors contribute to regulate aromatase expression in amygdala neurons before the critical period of brain masculinization. Our findings propose a novel mechanism orchestrated by sex chromosomes in which ERβ (but not ERα or AR) acts as the final effector in the action of E2 and DHT on aromatase expression in developing amygdala neurons.7.The embryos were obtained from CD1 mice raised in the Cajal Institute  Figs  and 7 an7. The medium was phenol red-free Neurobasal supplemented with B-27, N-2 and GlutaMAX I (Invitrogen). After 3 DIV the culture medium was replaced for 2 h by fresh medium devoid of supplements for Western blotting and quantitative real-time polymerase chain reaction analysis. Neuronal cultures were treated for 2 h with the following test compounds alone or in combination: E2 , DHT , 3β-diol , the selective ERα agonist propylpyrazoletriol , the selective ERβ agonist dyarilpropionitrile , the GPER agonist G1 , the selective ERβ antagonist 4-pyrimidin-3-yl]phenol  and the selective AR antagonist flutamide .E15 mouse embryos were classified according to the sex or/and genotype. Cultured amygdala neurons were prepared and maintained as describedCyp19a1, Ar, Esr1 and Esr2 gene expression were determined using SYBR Green Real-Time PCR in the Step OneTM Real Time PCR System (Applied Biosystems). The efficiency of the amplification was established using a calibration curve (5-point 1:3 dilutions) and the dilution used for the samples was 1:3 from the initial concentration. All primer-pairs showed a single product in the melting curve analysis and efficiencies [E = 2 ± 0.1 (E = 10[−1/slope])] between 90 to 110% (Table Cyp19a1 (E = 2.15) that was analysed according to Pfaffl49 .Total RNA was isolated from neuronal cultures with TRIZOL reagent (Invitrogen) as directed by the manufacturer and quantified using NanoDrop spectrometer (Thermo Scientific). The RNA was reverse-transcribed into cDNA using random primers (Biodynamics) and SuperScript II reverse transcriptase (Invitrogen). 4). Aliquots of each sample were used for total protein quantification according to Lowry et al.50. The samples were homogenized, combined with an equal volume of Laemmli’s buffer , boiled at 100 °C for 5 min, and centrifuged at 10,000 g for 15 min. Protein samples (20 μg/lane) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes . Rabbit anti-aromatase antibody (diluted 1:1000) is directed to amino acids 488–502 of mouse aromatase51. The aromatase antibody resulted in the staining of two bands, including a band at 51 KDa corresponding to the molecular weight of aromatase52. Secondary antibody conjugated to horseradish peroxidase  was used for the detection by enhanced chemiluminescence on X-ray film. After aromatase antibody blots were stripped by washing 10 times for 5 min each with Tris-buffered saline (TBS)-Tween (0.1%) at room temperature, 30 min at 55 °C with stripping buffer , and finally 10 times for 5 min each with TBS-Tween at room temperature. The stripped blots were then reproved with a monoclonal antibody against β-actin  as loading control. The resulting film samples were scanned and analysed with the ImageJ program (freely available at imagej.nih.gov).Western blotting was performed on homogenates of amygdala neurons raised in culture medium immediately after cell treatment. Cultures were washed twice with PBS at 37 °C and scraped with a rubber policeman into Ripa’s buffer , 50 mMTris, pH 7.5) with protease inhibitors  or three-way ANOVA . The loci of significant interactions were further analysed by Fisher’s least significance difference (LSD) post-hoc. When there were only two experimental groups, data was analysed using the Student’s t-test. A level of p < 0.05 was considered statistically significant. The n for statistical analysis was the number of independent neuronal cultures and corresponds to the number of pregnant mothers in CD1 cultures. For cell cultures prepared from FCG mice the number of independent neuronal cultures corresponds to the number of embryos of each genotype and treatment. The FCG embryos were obtained from 5 pregnant mothers.


Oryza sativa L, but may also increase tolerance . Through a series of greenhouse, screenhouse and field experiments, this study examines fitness  of the brown planthopper, Nilaparvata lugens (Stål), on resistant (IR62) and susceptible (IR22) rice varieties and age-related rice tolerance to planthopper damage under varying resource (nitrogenous fertilizer) availability. Planthoppers reared on IR62 in the greenhouse had lower fitness than planthoppers on IR22. IR62 became increasingly resistant as plants aged. IR22 was generally more tolerant of planthopper damage, and tolerance increased in IR22, but declined in IR62, as the plants aged. Rice plants infested at pre-tillering stages (3–4 leaf stage) in the screenhouse had greater losses to root, shoot and grain yield per unit weight of planthopper than plants infested at tillering stages, particularly in IR22. These trends were mainly due to the impact of planthoppers during pre-tillering stages and the length of exposure to the planthoppers. High nitrogen compromised IR62 resistance, particularly in tillering plants in the greenhouse study; however, high nitrogen did not increase planthopper biomass-density on IR62 in greenhouse or field cages. Tolerance to damage in IR62 at mid-tillering stages declined under increasing levels of nitrogen, but nitrogen increased tolerance during late-tillering stages. Planthopper damage to IR22 in field cages was severe and hopperburn (plant death) occurred in 83% of IR22 plants under high nitrogen (60–150 kg N ha−1). In contrast, despite planthopper infestations, damage to IR62 was low in field-grown plants and productivity  increased in IR62 under increasing nitrogen. Our results indicate that, whereas nitrogenous fertilizer increases planthopper fitness on susceptible and resistant varieties, the net effects of high nitrogen on IR62 include decreased planthopper biomass-density  and higher tolerance to damage during later growth stages .High resource availability can reduce anti-herbivore resistance (a plant’s ability to defend against herbivores and reduce damage) in rice,  It appeared that the brown planthopper, which is often regarded as the most serious pest of rice in Asia, is able to pre-empt extra available nutrients from the plant phloem before these can be used by the host plant – thereby decreasing plant tolerance under high nitrogen . Since plants face distinct challenges as they grow and develop, they are predicted to shift their defence strategies to counter their most probable biotic stresses . ResistaIn the present study, we examine the integrity of rice resistance under a range of nitrogen levels through a series of greenhouse, screenhouse and field experiments. We also examine how nitrogen affects ontogenetic changes in resistance and tolerance to planthoppers during crop development. In our experiments, we compared herbivory on the resistant rice variety IR62 against herbivory on the susceptible variety IR22. We used IR62 in our experiments as a modern variety with noted and consistently high resistance to planthoppers from recent screening in South and South East Asia , and bec22.1Bph1, bph2, bph5, bph7, bph8, Bph18, BPH25 and BPH26 (Nephotettix virescens (Distant), were most abundant.We focused our experiments on the brown planthopper, which is one of the most damaging rice pests in Asia . In our nd BPH26 . The ins2.2Bph32, Bph3(t) and BPH26 [a synonym with bph2]: Bph32 and BPH26 genes have recently been cloned (Bph3 locus or Bph32 gene (and not BPH26)  as resistance donors (Bph32 plays a role in inhibiting feeding by the planthopper (We used the resistant rice variety IR62 in our experiments. IR62 is one of few modern rice varieties with demonstrated and consistent resistance to planthoppers from recent greenhouse and field studies . There in cloned . It is s6) e.g., . Althoug6) e.g., . In conte donors . Furthernthopper . Althougnthopper . We used2.3We conducted a series of experiments to examine how the phenological stages at which plants are infested affect resistance and tolerance to the brown planthopper under gradients of added nitrogenous fertilizer. These experiments were conducted under greenhouse, screen house and field conditions. Greenhouse experiments are useful to compare planthopper fitness responses  to resistant and susceptible plants under low herbivore pressure. In greenhouse bioassays, severe damage to the plants, which would confound response estimates, is avoided because only small numbers of nymphs or adults are normally used. Using plants grown at staggered intervals, such short-duration bioassays can be used to estimate ontogenetic changes in resistance and tolerance. High herbivore densities are often required to examine responses by plants to sustained herbivory (over multiple plant stages and herbivore generations). In such long duration bioassays, plants can become rapidly pot-bound ; therefo2.3.1−1. The amounts of fertilizer added to the pots were calculated based on the estimated weight of top soil in a hectare of paddy (2000 t), the weight of soil in the pots (1.3 kg for size-6 pots), the proportion of nitrogen in the fertilizer (0.21 for ammonium sulphate), and the desired application rate. The final weights of ammonium sulphate were 0.186 g pot−1 to simulate 60 kg N ha−1 and 0.464 g for 150 kg N ha−1. A third group of control pots received no fertilizer. Seed was initially germinated under humid, dark conditions. After 3 days, healthy seedlings . For each bioassay, 54 size-6 pots (15 × 15 cm: height × diameter [H × D]) were filled with paddy soil. These were divided into three groups of 18 pots. Two groups of pots were treated with basal applications of ammonium sulphate to simulate field applications equivalent to 60 or 150 kg N haHoneydew production was examined by infesting plants of each age and at each nitrogen level with two pre-starved (48 h) gravid females in specially prepared feeding chambers. The plastic chambers confined the adults to within 5 cm of the base of the plants and were placed over filter paper, neatly fitted around the plant shoot. The filter papers had been treated with bromocresol green to indicate the nature of the honeydew as coming from the phloem (basic) or xylem (acidic) . After fTo examine nymph survival and development, 10 newly emerged nymphs were placed on rice plants (two plants per cage) of each age and under each nitrogen treatment. The plants were covered with acetate insect cages  with mesh widows for ventilation. Nymphs were allowed to feed and develop for 10 days after which the number of survivors and their developmental stages were recorded. The insects were collected at the end of the experiment and the plants cut above the soil level. All materials were dried at 60 °C in a forced draught oven and weighed.In a no-choice oviposition experiment, gravid females (one per plant) were placed on plants of each variety, at each plant age and under each nitrogen treatment. The plants were covered with acetate insect cages (as described above). Females were allowed to feed and lay eggs for 7 days after which they were removed and the plants dissected to count the eggs. The plants were cut above the soil and were dried in a forced draught oven at 60 °C before weighing.2.3.2−1) or at 60 kg N ha−1 and 150 kg N ha−1. Nitrogen was added to the pots as ammonium sulphate. The amounts of fertilizer required were calculated for size-10 pots that contained 5.5 kg of soil. Equivalent weights were 0.786 g of ammonium sulphate to simulate 60 kg N ha−1, and 1.964 g for 150 kg N ha−1. Two thirds of the required fertilizer was applied prior to transplanting and the remainder applied at the tillering stage. Soil in the pots was maintained at saturation by daily watering, which buffered against temperature extremes. Plants were sown at one time and infested with planthoppers at 15, 30 or 45 DAS . To initiate infestations under similar densities of attack, gravid females were placed on the rice plants according to the biomass of the plants at each infestation date. Prior to infestation, two extra control plants of each variety were destructively sampled, dried in a forced draught oven and weighed to estimate shoot biomass. The gravid females were applied to the plants at a rate of one planthopper per gram of plant dry weight.We evaluated the responses by IR62 and IR22 plants under three nitrogen levels when attacked by planthoppers at one of three plant ages. The screenhouse was covered by 1 mm mesh that restricted arthropod movement, but maintained normally cooler temperatures than in the greenhouse . The IR62 and IR22 plants were grown in size-10 pots (22 cm × 24 cm: H × D) under zero-added nitrogen (0 kg N haThe planthoppers and plants were contained in mesh cages (160 × 22 cm: H × D). A set of corresponding, control (non-infested) plants was also planted and maintained under mesh cages until harvest (≥85% grain maturity). Plants were monitored daily and pots were watered and weeded as necessary. Plants were not treated with any pesticides. Severely planthopper-damaged plants were destructively sampled before final wilting. The planthoppers were first collected from the plants using a vacuum sampler  and were then dried, counted and weighed. Plants were then carefully pulled along with their roots and were washed, dried and weighed. Where plants had reached physiological maturity , 60 kg N ha−1 and 150 kg N ha−1. The nitrogen treatments were replicated in six fields, with each field containing three plots . Separate sub-irrigation channels were installed around each plot. These connected to the main field canals for irrigation and drainage but prevented leakage of nutrients among adjacent fields or among plots within each field.A field study was conducted at IRRI Experimental Field Station  during the 2011 wet season (WS) and the 2012 dry season (DS). The site had deep clay soils with about 4% organic matter. The experiment was conducted in a series of 18 rice plots of 33 × 12.5 m (L × W) that were each treated with one of three nitrogen regimes. These were zero-added nitrogen  were installed in each of the plots. The cages were constructed of wooden frames and covered with 0.5 mm-bore insect netting. In June 2011 (wet season), IR62 rice plants were transplanted to two of the cages and IR22 to the remaining two cages as nine plants (hills) per cage arranged as three rows of three hills, each separated by 20 cm (hill spacing = 20 cm × 20 cm). The same set-up was repeated in 2012 (dry season) with transplanting conducted in January. Small plots such as these are considered adequate to assess yield and other plant characteristics in experiments such as ours where all plant genotypes used in the experiment have similar growth and development  .Thirty days after transplanting (mid-tillering stage), plants inside two of the mesh cages (one with IR62 and one with IR22) in each plot were each infested with 25 second-instar planthopper nymphs (225 nymphs per cage). The remaining cages were left as non-infested controls. Plants were allowed to grow and develop until harvest, or until they showed symptoms of heavy planthopper damage (noted by severe yellowing of the leaves). Planthoppers inside the cages were collected when the plants were heavily damaged and before severe ‘hopperburn’ had killed any plants. Planthoppers were collected using a blow-vac suction sampler . At the same time that insects were collected, a single rice plant was randomly sampled from the same cage. In the remaining cages (control and surviving infested), planthoppers were collected at the time of harvest (≥85% grain maturity) and a single rice plant sampled at the same time.After sampling, the numbers of tillers on each plant was recorded. The plants were then cleaned and divided into roots, shoots and panicles and the number of filled and unfilled grains recorded. All planthoppers and leafhoppers were separated from the complete arthropod samples. The planthoppers and leafhoppers were identified and dried separately (by species) in a forced draught oven at 60 °C for 3 days before being weighed. Plants, separated as roots, shoots and grain were also dried and weighed.2.4The effects of variety, plant age, nitrogen level and their interactions in the greenhouse experiment were examined using general linear models (GLM) with xylem-feeding, survival, planthopper biomass, biomass density, plant biomass change (per mg of planthopper), and eggs laid as dependent variables. Xylem-feeding was examined as a proportion of total honeydew production to standardize for planthopper biomass and different planthopper feeding rates . The covPlant growth parameters  recorded from control plants at the time of harvest in the screen-house experiment were examined using GLM (model = variety + nitrogen + interaction). Parameters for the infested plants were similarly examined, but included the factor ‘time of infestation’. We estimated the impact of planthopper populations on shoot biomass, root biomass and grain yield in their respective plants as ‘biomass change’ . Biomass changes were analysed using two-way GLM.Plant parameters  and herbivore parameters  from the field experiments were examined using split–split plot GLMs with nitrogen as the main plot and variety and infested/control treatments as subplots. Root, shoot and grain biomass changes and changes in tiller number as a function of planthopper biomass were calculated as above (biomass change). Due to natural infestation of the control plants, planthopper biomass was calculated as the difference between the biomass of planthoppers in the infested cages and the biomass in corresponding control cages for each variety, nitrogen level and season. This assumes that brown planthoppers had the greatest overall effect on the rice plants and that whitebacked planthoppers and green leafhoppers had similar individual effects with brown planthoppers on the plants. We believe that this latter assumption would have little consequence to the overall results because of relatively low numbers of the latter two species in the infested cages. Biomass changes were not estimated for IR22 plants because plants often had severe yellowing or ‘hopperburn’ (plant desiccation and death due to planthopper feeding) soon after insect sampling and consequently high levels of intraspecific competition and declining planthopper fitness that prevented meaningful estimates of tolerance. Therefore, biomass changes were examined only for IR62 using univariate GLM with nitrogen as the independent variable and removing the effect of fields (blocks).Residuals were plotted following all analyses. Where data were not normal and homogenous we applied appropriate transformations (indicated together with the results below). Where data could not be normalized we ranked cases. Analyses were conducted using SPSS v.22 .33.1−1), but higher excretion of xylem-derived honeydew by planthoppers on IR62 under low nitrogen levels (0 and 60 kg N ha−1) – indicating an improvement in feeding efficiency , declined under increasing plant age  and under increasing nitrogen  . A significant [nitrogen × plant age] interaction for biomass density  was due to lower densities on 30 and 45 DAS plants under high nitrogen, but similar densities across nitrogen levels for 15 DAS plants (Planthopper biomass density (which indicates the net effects of nitrogen on planthopper-infestations under varying host plant conditions) was lower on IR62 (F= 0.037) A–C. The S plants A–C. All 1,90 = 2.662, P ≤ 0.001) suggesting that IR22 had higher tolerance to planthopper feeding in these short duration bioassays. Planthopper impact declined on IR22 plants as these aged, but increased on IR62 with age producing a significant [variety × plant age] interaction . There was also a significant [nitrogen × plant age] interaction  because high nitrogen increased tolerance in 45 DAS plants, but did not significantly affect tolerance in 15 and 30 DAS plants , shoot biomass  and root biomass  was consistently higher on IR22 compared to IR62  and roots  per mg of planthopper were consistently lower where plants had been infested at tillering stages  and root biomass loss  because of similar losses to plants of both varieties at older plant stages, but higher losses to IR22 plants infested at 15 DAS (pre-tillering) compared to IR62 plants infested at the same age . Further details of the planthopper and leafhopper densities are presented in Supplementary Fig. S1.In the field experiments, IR62 had a higher number of tillers and a higher proportion of filled grain and attained greater grain yields than IR22 , Table 4−1: Although non-infested plants also had natural colonisation by planthoppers, the densities were similarly low on both IR62 and IR22 in the non-infested treatment   and interactions were not significant . Plants infested with planthoppers had lower tiller numbers, lower root and shoot biomass, and yielded less grain  A,B. Biomss grain , Table 4ss grain , Table 4ss grain , Table 42,15 = 3.401, P = 0.061, linear contrast P = 0.021: 2,15 = 2.370, P = 0.128, linear contrast P = 0.048: 2,15 = 4.330, P = 0.033, linear contrast P = 0.010: 2,10 = 0.099, P = 0.906, linear contrast P = 0.725: 2,15 ≥ 0.110, 0.062 ≥ P ≥ 0.896).During the 2011 wet season, planthopper populations on IR62 were low and the effects of planthoppers in reducing tiller number  and in tolerance on both IR22 and IR62 . Furthermore, nitrogen clearly reduced resistance in IR62 and reduced tolerance in young IR62 and IR22 plants (15–30 DAS) but increased tolerance in older IR62 plants (45 DAS). Infestations of rice plants by planthoppers, and other herbivores, are not restricted to discrete life stages; indeed planthoppers can have up to three generations on a single rice plant . Our scrExperiments using potted plants have been noted elsewhere as limited in their usefulness for making predictions about field situations, particularly where fertilizers are included as a treatment . We tookHigher tolerance to planthopper feeding at reproductive stages − including during grain-filling, will be particularly beneficial to rice farmers. During the last two decades, tolerance as a concept in herbivory has gained increased attention. However, several confounding concepts of tolerance exist, including tolerance as a component of resistance i.e., , and tol4.2Bph3 locus had an anti-feeding effect on planthoppers. In feeding tests, these authors found mortality of the planthoppers to increase as schaftoside concentrations increased from 250 to 500 μg mL−1. More recently, Bph32 gene (from PTB33) encodes a protein similar to lectins that act on insect glycoproteins or tissues and inhibit insect feeding. The gene was highly expressed in the leaf sheaths of rice at 2 and 24 h after planthopper infestation, suggesting an induced response pathway . As these insects grow and develop, their damage potential increases, thereby representing a greater threat to older plant stages. Furthermore, early-tillering rice plants can often compensate well for herbivore damage by rerouting nutrients among tillers or by producing new tillers .We expected that older IR22 and IR62 plants (at low nitrogen levels) would tolerate planthoppers by continuing to route nutrients to reproductive organs such as panicles and grain . In our 4.4In our experiments, planthopper fitness often increased on high nitrogen plants, particularly during late tillering stages (45 DAS). Recent studies have indicated that anti-herbivore resistance associated with a range of different resistance genes is compromised by high nitrogen conditions . In the 4.5Bph3 locus or the closely related bph4 locus are no longer effective against the brown planthopper even in the Philippines −highlighting the role of associated rice genes and variety traits in enhancing resistance  can only be considered as a component of integrated pest management and that a greater emphasis on ecological studies will be necessary to successfully deploy resistant rice varieties in modern rice production systems. Understanding not only resistance, but also tolerance to herbivores under varying crop management practices will help design improved crop production systems that support the natural regulation of crop pests.Our results indicate that farmers using high fertilizer applications on IR62 will not appreciably accelerate adaptation by planthoppers to the variety’s specific resistance genes. However, because IR62 is naturally infested by planthoppers in the field (as noted in our field experiments), there is potential for planthoppers to adapt to the variety see  and by asee also . Further


By changing the linker unit between the NBD units, we can at the same time fine-tune light-harvesting and energy densities of the dimers and trimers so that they exceed those of their monomeric analogs. These new oligomers thereby meet several of the criteria to be met for an optimum molecule to ultimately enter actual devices being able to undergo closed cycles of solar light-harvesting, energy storage, and heat release.Molecular photoswitches can be used for solar thermal energy storage by photoisomerization into high-energy, meta-stable isomers; we present a molecular design strategy leading to photoswitches with high energy densities and long storage times. High measured energy densities of up to 559 kJ kg Molecular solar thermal systems are promising for storing solar energy but achieving high energy storage densities and absorption characteristics matching the solar spectrum is challenging. Here the authors present a design strategy for electronically coupled photoswitches which allow for high energy density storage for solar energy storage applications. Molecular solar thermal (MOST) systems represent a promising avenue for harvesting and storing solar energy. In this approach a molecule is converted by photoisomerization into a higher-energy isomer, which is capable of storing the energy until released by a heat trigger or catalyst, converting the meta-stable isomer to the original light-harvesting isomer10. Norbornadienes have shown to be a promising candidate for MOST due to the high energy difference between the norbornadiene (NBD) and quadricyclane (QC) photoisomer of approximately 96 kJ mol−1 , leading to a higher energy of isomerisation of the second switching event (QC−NBD to QC−QC). Simultaneously having a shared donor reduces the molecular weight per norbornadiene unit. We chose to synthesize two series of dimers as well as one trimer of NBD that resemble the previously synthesized donor−acceptor monomers 1 and 2, with absorption onsets of 358 and 374 nm, respectively14.The idea for the present work was to engineer the stability of the high energy photoisomer by having two electronically coupled photoswitches with separate barriers for thermal conversion Fig. . The rea3 as substrate14. The simplest dimer was synthesized by Sonogashira coupling with trimethylsilylacetylene, affording 4 in a yield of 63%. The silyl protecting group was removed with potassium carbonate in THF/MeOH, and the resulting terminal alkyne was subjected to a second Sonogashira coupling with 3 to afford the acetylene-linked NBD dimer 5 in 47% yield. In the second Sonogashira reaction, standard conditions using phosphine ligands did not give any of the wanted products due to instability of the terminal acetylene. Instead, the Pd2dba3/AsPh3 catalyst system26 was used as it improved the reaction rate. Utilizing the same reaction conditions as for the synthesis of 4 with either 1,3- or 1,4-diethynylbenzene (6 or 7) gave the two NBD dimers 8 and 9 in 39 and 31% yield, respectively. Similarly, with 1,3,5-triethynylbenzene 10 as substrate, a triple Sonogashira coupling furnished 11 in a yield of 24%.The synthesis of NBD dimers and trimers is shown in Fig. NBD units directly linked to a phenylene spacer was synthesized benzenes 12 and 13, respectively, with cyclopentadiene (under microwave heating) affording the dimers 14 and 15 in yields of 77 and 45%.To further investigate the effect of the spacer group, a second series with two zed Fig. . Initialmeta and para isomers. The systems that are para substituted  give the highest onset of absorbance, which can be explained by the linear conjugation pathway between the two NBD chromophores, providing a larger conjugated system compared to the meta substituted compounds. The spectra of the meta substituted dimer 9 and the trimer 11 have very similar absorption onsets; yet, the trimer with three NBD units has a higher molar absorptivity. Interestingly the spectra are also similar to that of the corresponding monomer 2 , the dimers and trimer could all be fully converted to the corresponding QC−QC dimers, data shown in Table QC−NBD form by irradiating around the onset of absorption. Switching to the QC−NBD could be achieved for compounds 5 and 14 by irradiation at 405 nm. In addition the switching events were also studied by NMR spectroscopic analysis. For compound 5 it was found that irradiation at 405 nm forms a thermal-photostationary state between 5NBD−NBD and 5QC−NBD, which is dependent on the intensity of the lamp and the temperature of the sample, due to fast back conversion of 5QC−NBD (vide infra). The ratio found from NMR studies was 54:46 . For compounds 8, 9, 11, and 15 the quantum yields are calculated based on the number of photoconversion events, taking into account that two or three NBD subunits are present per oligomer , was determined from an Arrhenius analysis , until fully converted; the solutions were concentrated by a stream of nitrogen and the resulting QC subjected to differential scanning calorimetry (DSC) (Table 11 and 14 partly decomposed at the temperatures of the DSC experiment (>140 °C), so the energy densities of these compounds are not included. Isomerization of 5 in either CDCl3 or toluene-d8 for DSC experiments (~1 M) could not be achieved by irradiation at either 365 or 340 nm, possibly due to lower quantum yields in these solvents as well as the short lifetime of the QC−NBD form (minutes) making quantitative conversions impractical. The heat release data for QC−QC dimers of 8, 9, and 15 are listed in Table 20. The DSC sample for 9 was a mixture of 9QC−QC and 9QC−NBD, which results in a large uncertainty, even though a correction taking this into account was performed. This is probably the reason for the large difference between calculated and measured values.To obtain the energy densities of the high-energy C) Table . Compounpara phenylene-bridged NBD dimer 14, conversions between isomers occurred stepwise—both in the forward and backwards direction—and with significantly different NBD absorption maxima for the NBD−NBD and NBD−QC states. Our molecular design demonstrates the attractive feature of blueshifting the NBD absorption after the first NBD-to-QC photoisomerization as it ultimately furnishes a very energetic QC−QC state after the second photoisomerization as well as a prolonged storage time. As the first energy discharge (QC−QC to QC−NBD backreaction) is the rate-determining step, full discharge of the system will conveniently be promoted by triggering this event. The system contrasts in this respect related dihydroazulene-vinylheptafulvene dimers incorporated in macrocyclic structures for which instead the first discharge is fast and the second slow29.In conclusion, we have developed efficient synthetic protocols for the preparation of high-performance norbornadiene dimers and trimers. The quantum yield of photoconversion is near quantitative, up to 94% per NBD subunit. For the −1 (257 Wh kg−1) and measured densities up to 559 kJ kg−1 (155 Wh kg−1) greatly exceed the original targets of 300 kJ kg-115 highlighting the potential of applying molecular photoswitches in future solar thermal energy storage technologies. As the strongly coupled dimers 5 and 14 show a higher barrier for the first step in the thermal conversion compared to the second thermal conversion, this molecular motif can be used in future design of high-performance molecular switches for solar thermal energy storage as well as other applications. In future work a donor group could be attached to the benzene ring to give an enhanced redshifting of the absorbance spectra, or changing the benzene ring to more electron-rich aromatics. Another interesting approach would be to have two NBD units directly linked, in an attempt to increase the multimode switching by a greater coupling between the NBD subunits.The calculated energy densities of the dimer and trimer systems of up to 927 kJ kgCyclopentadiene was distilled by cracking dicyclopentadiene over iron filings and stored at −80 °C, prior to use. Tetrahydrofuran used for Sonogashira coupling reactions was distilled over a sodium/benzophenone couple. All other chemicals were used as purchased from commercial sources. Purification of products was carried out by flash chromatography on silica gel . Thin-layer chromatography was carried out using aluminum sheets precoated with silica gel.3 onto an ATR attachment, where relative intensities are denoted as vw (very weak), w (weak), m (medium), s (strong), and sh (shoulder). All melting points and heat release of neat quadricyclanes were recorded on a Mettler Toledo DSC 2 apparatus. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on a Varian 400 MHz instrument or a Bruker 500 MHz instrument with a noninverse cryoprobe using the residual solvent as the internal standard . All chemical shifts are quoted on the δ scale (ppm), and all coupling constants (J) are expressed in Hz. All solution-based spectroscopic measurements were performed in cyclohexane in a 1-cm path length cuvette on either a Cary 60 or a Cary 100 UV−Vis spectrophotometer, scanning the wavelength from 600 to 200 nm coupled with Peltier temperature control. Photoswitching for wavelengths 310, 340, 365 and 405 nm were performed using Thorlabs LED lamps M310L3, M340L4, M365F1 and M405LP1 respectively. The thermal back reaction was performed by heating the sample (cuvette) by a Peltier unit in the UV−Vis spectrophotometer. Quantum yields were measured by the published procedure28 using a high concentration regime (absorption above 2 at the irradiation wavelength) using potassium ferrioxalate and tris-phenanthroline iron (II) complex as a chemical actinometer and Thorlabs LED lamps M310L3, M340L4 and a fiber-coupled LED (M365F1) respectively for irradiation. Irradiation was done perpendicular to the cuvette in a fixed setup approximately 5 cm distance between LED and sample, with stirring, ensuring no movement of the lamp during the experiment. Mass spectra (HRMS) were either acquired by electron spray ionization (ESI) using an Agilent 1260 Infinity fitted with an Agilent 6120 quadrupole for or an ESP-MALDI-FT-ICR spectrometer equipped with a 7 T magnet . Elemental analyses were performed at London Metropolitan University. Compounds 314, 1227, and 1327 were made by their respective literature methods.Infrared (IR) spectra recorded on a Perkin-Elmer Frontier FT-IR instrument as films evaporated from CDCl31. All norbornadiene and quadricyclane compounds were fully relaxed. The storage energy was computed as the energy difference between the norbornadiene and quadricyclane forms.Calculations for the heat release from QC to NBD were performed at the B3LYP/6-311+G* level  using the NWChem packageData supporting this study can be found in the Supplementary Information file. Upon request to the corresponding author, further information supporting this study can be made available.Supplementary Information