We have reconstituted a eukaryotic leading/lagging strand replisome comprising 31 distinct polypeptides. This study identifies a process unprecedented in bacterial replisomes. While bacteria and phage simply recruit polymerases to the fork, we find that suppression mechanisms are used to position the distinct eukaryotic polymerases on their respective strands. Hence, Pol ε is active with CMG on the leading strand, but it is unable to function on the lagging strand, even when Pol δ is not present. Conversely, Pol δ-PCNA is the only enzyme capable of extending Okazaki fragments in the presence of Pols ε and α. We have shown earlier that Pol δ-PCNA is suppressed on the leading strand with CMG . We propDOI:http://dx.doi.org/10.7554/eLife.04988.001 Cells must replicate their DNA before they divide so that the newly formed cells can each receive a copy of the same genetic material. DNA replication requires complex molecular machinery called a replisome, which comprises multiple proteins, enzymes, and other molecules. First, an enzyme called a helicase starts to unwind the double-stranded DNA into two single strands. This process continues while other enzymes, called polymerases, use the exposed single strands as templates to make complementary new strands of DNA. One of these new strands is built continuously and called the ‘leading strand’. The other newly forming strand—the ‘lagging strand’—is made in the opposite direction, as a series of short fragments that are later joined together.The replisomes in bacterial cells have been well studied, but many researchers are investigating the composition of the replisome in animals, plants, and fungi . Now, Georgescu et al. have essentially rebuilt a eukaryotic replisome from 31 different proteins in a test tube and confirmed that it can make both leading and lagging DNA strands—just like in a normal cell. Further experiments revealed that the polymerase enzyme that operates on the leading strand cannot work on the lagging strand and vice versa. This exclusivity is unique to eukaryotic DNA replication, as bacterial polymerases can use either DNA strand as a template.Georgescu et al. then found that the eukaryotic polymerases are actively prevented from copying the ‘wrong’ strand of DNA and suggest that the helicase enzyme that unwinds the DNA might be behind this activity. Important future studies must now address how the replisome deals with obstacles created by certain DNA-binding proteins and damaged DNA and how it interfaces with the molecules that control cell division and DNA repair.DOI:http://dx.doi.org/10.7554/eLife.04988.002 Composition of the eukaryotic replisome and the function of its various proteins is an area of active investigation. Cellular studies reveal that eukaryotes use two different DNA polymerases for the leading and lagging strands, Pols ε and δ, respectively . PrimingWhile in vitro synthesis of the leading strand replisome has been accomplished with the purified CMG complex from budding yeast , the disStudy of the eukaryotic replisome identifies a new process that has no precedent in bacterial systems. Bacteria use simple recruitment processes to attract and hold polymerases to the fork. These are typically mediated by polymerase interactions with other proteins at the replication fork, such as the helicase and sliding clamps . However32P-dCTP (leading) or 32P-dGTP (lagging).We expressed and purified yeast CMG helicase in our previous studies and demonstrated its function in leading strand synthesis with Pol ε . The disEscherichia coli. A mixture of cells containing the 4 subunits were lysed, and Pol α was purified as an intact 4-subunit holoenzyme . The analysis also confirms that Pol α cannot perform priming and extension in the absence of CMG (lanes 1–3) and that ϕ29 Pol cannot perform lagging strand synthesis (lanes 13–15).To determine if Okazaki fragments are distributed over the length of the DNA template, we used tive gel . The anaPol ε is the leading strand enzyme and presumably takes over the leading strand from Pol α after this distributive enzyme dissociates from DNA. To determine if the Pol α/ε switch occurs as expected, we preloaded CMG on the forked DNA, then added increasing amounts of Pol α either with or without Pol ε, and stopped the reactions after 20 min. If Pol ε takes over the leading strand from Pol α, full-length products will be observed sooner in reactions that contain Pol ε because in the presence of CMG, this enzyme synthesizes DNA faster than Pol α. The results show full-length product in all the lanes containing Pol ε with Pol α .In order to compare total leading and lagging strand synthesis rates, we setup standard replication reactions by loading CMG onto the nucleotide-biased forked DNA, followed by the addition of all three DNA polymerases in the presence of RFC and PCNA; the reactions were divided, and replication was initiated by the addition of either 32P-dCTP (leading) or 32P-dGTP (lagging) in the three polymerase replisome system shows similar amounts of leading and lagging strand synthesis . Indeed, polymerase recruitment by binding clamps and the helicase underlies attraction of polymerases to replication forks of bacteria, its phages, and the SV40 virus. Recruitment is also partly responsible for polymerase placement in eukaryotes. For example, Pol ε binds directly to CMG helicase, stabilizing it on the leading strand . HoweverWe were surprised to find that Pol α polymerase activity is highly functional with CMG on both leading and lagging strands in the absence of other polymerases. Pol α lacks the high fidelity of Pols ε and δ and does not provide bulk leading or lagging strand synthesis in cells, and thus processes must exist that suppress the polymerase activity of Pol α. In fact, we find many ways that Pol α polymerase is suppressed. One mechanism is by Pol ε positioning on the lagging strand. Interestingly, Pol ε is also suppressed on the lagging strand; perhaps, Pol ε is suppressed from function on the lagging strand by the relative orientation in which CMG holds Pol ε. On the leading strand, Pol ε simply prevents Pol α polymerase extension by switching with it and becoming processive with CMG. This can be likened to the switch of Pol δ with Pol α that prevents leading strand synthesis by Pol α in the SV40 system . Pol α pThe exclusion processes that underlie eukaryotic fork leading/lagging strand function are summarized in E. coli Pol III replicase  complex, directing the single Pol ε molecule to the leading strand. Suppression of Pol α by Pol ε on the lagging strand could perhaps be explained by competition of these polymerases for CMG, where Pol ε is suppressed from extending the primer. In this connection, the Pol2 gene encoding the catalytic polymerase actually contains two polymerase structures: the active polymerase/exonuclease on the N-terminal half and B-family polymerase in the C-terminal half of Pol2 that is presumed to be inactive . One posNumerous proteins travel with the replication fork, and it is not possible to know a priori how many are needed for functional leading and lagging strand replication. This is the first study to reconstitute leading/lagging strand replication with three pure polymerases in a eukaryotic system. It reveals that many of the proteins that move with forks are not required to recapitulate leading/lagging strand synthesis in vitro. For example, Ctf4/AND-1 is essential in most cells (not in budding yeast), and yeast Ctf4 helps recruit Pol α bind to RPCs , yet theThe current report also reveals that Pol α can prime the leading strand directly. To our knowledge, before this report, all proposed models of leading strand initiation in bacteria and SV40 show priming on the lagging strand of a bidirectional origin, which becomes the leading strand of one fork, rather than directly priming the leading strands . We showCellular studies indicate that nucleosomes are involved in determining Okazaki fragment size , but datReconstitution of cellular replisomes in vitro should provide a framework to explore the effects of other proteins that move with forks and of post-translational modifications that control eukaryotic forks in response to the cell cycle and DNA checkpoint mechanisms. Reconstituted systems should also enable detailed study of factors that maintain the epigenetic state of a cell during replication.2, 5 mM imidazole, 20 mM KOAc, and 350 mM KCl. Buffer D is 25 mM Tris-OAc pH 7.6, 40 mM K-OAc, 40 mM K glutamate, 2 mM Mg-OAc2, 1 mM DTT, 20% glycerol, and 0.25 mM EDTA. Stop buffer is 1% SDS, 40 mm EDTA. Buffer H is 20 mM Hepes pH 7.5, 10% glycerol, 1 mM EDTA, 2 mM DTT, 350 mM KCl, 1 mM ATP, and 4 mM MgCl2.Radioactive nucleotides were from Perkin Elmer. Unlabeled nucleotides were from GE Healthcare. DNA modification enzymes and ϕ29 DNA polymerase were from New England Biolabs. DNA oligonucleotides were from Integrated DNA Technologies. Protein concentrations were determined using the Bio-Rad Bradford Protein stain and bovine serum albumin as a standard. Buffer A is 20 mM Tris-HCl, pH 7.5, 5 mM DTT, 0.1 mM EDTA, and 4% glycerol. Buffer B is the same as buffer A except 20 mM Tris-acetate, pH 7.5 was used in place of 20 nM Tris-HCl. Buffer C is 25 mM Tris-Cl pH 7.9, 10% glycerol, 1 mM DTT, 1 mM MgClE. coli SSB , a strain constructed from W303  . Pol12 was transformed into E. coli BL21(DE3)codon plus RIL , then induced with IPTG for 8 hr at 15°C. Pri1 and Pri2 were co-expressed in E. coli BL21(DE3) cells by IPTG induction for 8 hr at 15°C. A 12 l culture of induced yeast cells for Pol1 and 1 l of each induced E. coli cultures for Pol12 and Pri1 and Pri2 were co-crushed in a cryogenic mill as described for CMG (g for 1 hr at 4°C. Anti-Flag agarose (1.2 ml) was added to the supernatant (80 ml) and incubated with slow rotation for 1.5 hr at 12°C. Beads were collected by centrifugation at 1500×g, washed twice with 5 ml 50 mM Hepes pH 7.4, 250 mM potassium glutamate, 1 mM EDTA, then loaded into a gravity column. The column was washed with 15 ml 50 mM Hepes pH 7.4, 250 mM potassium glutamate, 1 mM EDTA, 10% glycerol, and Pol α was eluted with 5 ml 50 mM Hepes pH 7.4, 250 mM potassium glutamate, 1 mM EDTA, 10% glycerol containing 20 μg/ml Flag peptide. The eluent was diluted to a conductivity equal to 150 mM NaCl using 25 mM Hepes pH 7.4, 1 mM EDTA, and 10% glycerol, then applied to a 1 ml Heparin agarose column. Pol α was eluted with a gradient of 100 mM to 1 M NaCl in 25 mM Hepes pH 7.4, 1 mM EDTA, 10% glycerol. Peak fractions were pooled, aliquoted, and stored at −80°C. Typical yield of Pol α was about 3 mg.Proteins were purified as described: RPA , E. colicoli SSB , PCNA  . The Pol for CMG . Frozen To make the linear fork DNA substrate, a 3.2 kb sequence of DNA was designed such that one strand lacked dC residues and thus the other strand lacked dG. The G-rich strand was examined to eliminate runs of four G residues. The resulting 3260 bp sequence was synthesized by Biomatik . The first four and last two bases represent overhangs generated by BsaI and BtsC1, respectively. Both of these enzymes cut outside of their recognition sequences, and their recognition sequences are excluded from the template.CGGTATTCTAACCACATTAATCTACACCTCTCACACACTACTCATATCATCTTCCAAAACCCACCTTTAAAAAACCCTTTATCCACACTCATCACCATTTCACCAACCTTTTTCTTAATTCTACACAAATCCAATTAACCTATCTCCAATTTTAACTCCATCACCTCTTATATTAACCCACCTACTACTCCAACAATACCCTATCAAATCTACTTCTATCTCAAAACTATCACCTACTCCTTCCATCATAATCCACTCTTATCAATTAAACAATTATCCTTCTTTCCCACCATCACTCACCATCTTTTCTTAACACCCTTAACATTTCCTTTTATAAAACACTTCCAATCCTATTTTCTCACTATCCCACCCACCATAAAAACTATCTCACCCTAACTCAACCCTTTCCCTCTCACCAACACTCCTTTATCACACACACTTTACCACACAAATCCCTCCATCATACACCTTTACCCTCAAATCCTAAACCACCTAACTATTCCACACAATATATCTACAAAAATTTACTTTTCCACATCTCCAACCCTTCCAACACCTTAATCCCAAACCTTAACTAACCTCTCTTTAATACTTCCTCCCATTCCCACCACATACACCAAATTATCTTCAACTCAAAAACCTAAACTCTCCTTTTTATTCCCTATAAAAACTCTTAACCCTCCAATATACAACTCTAACTAACTTCATTATCAACCAATCTTCCTCTACTTCCTCATCTTATAATTTATCCATTCAAAATAACCTACCTACCACCTCTCCTCTTCACTTCCTACCCTAAAATCACCACCTTATCCCTAATTTACCTCTTTCAACTTTCCTTAACCCAACTTCTCAATCCTACTTCACTTACTTCTTATAAAACCATCATTATCACACTACACATTACTCTATCTATCCAATCATCACCTTCTACAATCCAAACTATCCCACTACCCTCTCATTCTACCTTTTCATCTATCTCAAACTATCCACCAATCCATACCTCAAACTTTAACCACCCACTCCAAATCTACAACATAAAATTAACCTATCACATTCCAAACTAATCACCCTAACCCTAACACCCTTTTATCCTCACCAAAATTACCATTTTCCTCTTTACTCATAAACAAACATTCTCACCCATTTATAAAACACTTAATACCCACTTAATTCACTTCCTTTTTCTACCTCACCATCATCAACTCCTAATATCAACAACCCAAAATCACCACCTATATCCTCATCTCCTATATAAAAAACTTCACATCTCAACCTCAAACCACCTATTCCACTTAATCCCAATCAACCTATCAACTCTACAACCTACTCTTCAAATACATCTCCTATCACTTTCCCACCTCCTTCAATCAATTATAACTTTATCACCTAAACATTCTAAAATCTCCTATCCACTACATCACATAAACCTAAACCTACTACCAACCTACCATTATCCTTATCAAACATCATCAATTCCATCTTTCTTTATACCCTCTCCATATCTCTCTCATTAAAAAAACCAAAATCTAACAACTTCTTATTTCTAATCAAAAAAACAATCAACCTAACTCATAAAATCTTCACCTTAAAATTCCTTTACATTTAACAAATCCACTCTCCCTATCTTTTTCATATCAAACTTATCTAAACCACTATCCTCATTTATCCTAATACTCCATATACAACACCAAATTTCTTATATCTCATCTAAAATCCTCCACCAATATAAACTCCTCTTTACCATTTCCACTCAACACACCAAATCTTATTATTCCATCAAATCTAATCCATTACCATCATCAACCCTAATAAACCTACTTCCCAACTTTTATCTCTCCACTACCACACCAAAAATTAACCTCCTCTAAAAACTATCATTCCCTTTACCTCTTCCACATTCCACCTATAACTCCTCATCTTAAAACCAATCAACACCAAACAACTATCTCACCATATTTCCTCTCCAAACCAACAAATTAACAATCCTACCCACTCCAACCTCCACATTACTAATAACTAAACTTACCTTACCTACCACACCCTATCAACCATATTTAAAAAATTACTTATTCACTAACTAAAACATCACCCACAAACTTAAATCATCACCTCCTCTTTTCCACCTTATTCACCAACCCAATCTATCTATCTCACCTATACCTTTCCCTAATATCTTTTACTAACCCTATAAATACCACAATTCTAAAAAACCCATACTTATCTCACACATCACTTTATACTTCACTCTTAAAATACCCTCCAATATATATTACAACCCAAAAATATCTCCCTTCTATCTCCTACACACAAATTATACCACTTTTAAACCACTCCTCATCTCTAACCCAACCCTCTACAATTCCATACATCTCTACTATCAACATCACTCCTTCTTTTCCACTCTTCTCTCCACATCTTTATTAAACATCTCCTCCTCATTTTCACATAACTATTTACTAAATAAATTTACCTAAACTACATTTATTAAAACCCTACAACATACTCCTTATTCTCCTACCTACCATTCTCTAATCTCTTTACATTCTACTACTTTCCTACCTACTATCTCAATAAATTCACTTTCCTTTCACCACACACCAACACACCTTCCTCCAAATTCTTTATATCTCCTTCTCCTAACCAAATTCCTCACTAATAACATCTTACCTCCCTACCTTTTTCCTTCTACCCTCCACCATTTCCCAACCTCATACTCAATAATCAATTTACCCTCCCACAACATAACTCTATTAACACCCATTTTCTATCCATCAACTTCCTATTACTTAAATTATCTTTTAAACCAATAAAACTCCACCTCAACCACCCACCTCTCTCTTTCAATCCAATTTCAATCTTTCCAACCATTCTATCTACCCTAAACTATTAACTATCTATCTCACCCACAATCCCTCCTACAATTCACAACAACATTCCACCACTTCACTTTATCTTCACCTCCAAACTTATTCCTTCCCATTATCACCTTCTCCAAAACCCACAAACATCTAACTCTCATCTCTCAACACTTTCTACCCATTCTCTCTAACAAAATTCCCTTACTCTTTATTCACAAAACCACTAAAATCACCACAACAACCCAACAAAAACAAAATCCTCACTTACCTATACTCAATAAATCCTTCAACTCATTATTCTATTTCTAACCCTAAATCAAAACTCCCATATCTACCATTCTTTCCACTCAATTAAATCCCACCAACCCTTATTTTCCTCCAATAACTTAACC.The synthetic 3.2 kb DNA was cut from the plasmid using BsaI-HF and BtsCI and purified from low-melting agarose gel. 35 pmol of the 3.2 kb linear DNA template was ligated to a fivefold excess of forked junction on the BsaI end and to a 10-fold molar excess of a short blocking duplex on the BtsCI end. To make the forked junction, 1050 pmol 1T oligonucleotide was annealed to 175 pmol 160mer oligonucleotide, as described . The 16032P-dCTP, while for lagging strand replications, we used 20 μM dGTP and 10 μCi 32P-dGTP. Exceptions to this protocol are noted in the figure legends. Timed aliquots were removed and quenched upon adding SDS and EDTA to final concentrations of 0.5% and 20 mM, respectively. Quenched reactions were analyzed in 0.7% or 2% alkaline agarose gels and imaged in a Typhoon 9400 PhosphorImager .Replication assays contained 1.5 nM linear forked DNA, 24 nM CMG, 400 nM RPA (unless noted otherwise), 20 nM PCNA (unless noted otherwise), 6 nM RFC (unless noted otherwise), and Pol α, Pol ε, and Pol δ as indicated in the figure legends, in 25 mM Tris-acetate pH 7.5, 10 mM Mg-acetate, 50 mM potassium glutamate, 5 mM DTT, 0.1 mM EDTA, 40 μg/ml BSA, 0.1 mM AMP–PNP, 5 mM ATP, 200 μM each rCTP, rUTP, rGTP, 60 μM of each unlabeled dNTP, and 20 μM of the labeled dNTP. Reactions were staged as follows. CMG was added first and pre-incubated with the DNA and 0.1 mM AMP-PNP for 10 min at 30°C, then the noted polymerases together with the RFC and PCNA (where indicated) were added, along with dATP, dCTP for an additional 2 min. Replication was then initiated upon the addition of a solution containing the RPA, ATP, dTTP, and dGTP. It is important to note that for leading strand replication reactions, we used 20 μΜ dCTP and 10 μCi 32P-dCTP (leading) or 32P-dGTP (lagging) was added. Timed reactions were stopped with an equal volume of 2× STOP solution (40 mM EDTA and 1% SDS) and spotted on DE81 filter papers, then analyzed using a Perkin Elmer Liquid Scintillation Analyzer . Separately, we performed control replication reactions using ϕX174 ssDNA coated with RPA, containing a known amount of Gs and Cs, confirm that 32P-dCTP and 32P-dGTP are equally incorporated by each of the DNA polymerases in our experimental conditions.In order to compare total DNA synthesis rates on leading and lagging strand, we performed standard replication reactions containing all three DNA polymerases at 10 nM, CMG (30 nM), RFC (10 nM), PCNA (20 nM), and RPA (400 nM) in presence of 1 mM ATP, rNTPs , and 30 μM dNTPs; reactions were divided, and either 32P-dGTP to label the lagging strand; the second reaction utilized 1.5 nM unprimed forked DNA and 10 nM each Pol ε and Pol α along with 20 μM dGTP and 10 μCi 32P-dGTP to label the lagging strand, and the third reaction contained DNA-primed forked DNA and 1 U of ϕ29 DNA polymerase along with 20 μM dCTP and 10 μCi 32P-dCTP to label the leading strand. Each reaction was quenched upon heating to 65°C for 10 min to inactivate the CMG and polymerases. Then, reactions were divided into three tubes, one was untreated, the second was treated with EarI, and the third was treated with PsiI, adjusting the reaction buffer for each enzyme with the commercial provided buffer. Reactions were analyzed in a 2% native agarose gel followed by autoradiography in a Typhoon 9400 PhosphorImager (GE/Molecular Dynamics).Three replication reactions were performed as described above with the following differences. The first reaction used 1.5 nM unprimed forked DNA and 10 nM Pol α with 20 μM dGTP as well as 10 μCi 32P) dTTP and incubated at 30°C. At the times indicated, 25-μl aliquots were removed and quenched by addition of an equal volume of 1% SDS/40 mm EDTA. Products were analyzed in 0.7% alkaline agarose gels. Gels were dried, exposed to PhosphorImager screens, and imaged using a Typhoon 9400 PhosphorImager (GE/Molecular Dynamics).Reactions contained 1.5 nM ϕX174 circular ssDNA (as circles) primed with a DNA 30-mer and pre-incubated for 10 min with 420 nM RPA (as heterotrimer) in 20 mM Tris-Cl (pH 7.5), 50 mM potassium glutamate, 5 mM DTT, 0.1 mM EDTA, 40 μg/ml BSA, 8 mM MgOAc, 0.5 mm ATP, 5% glycerol, and 60 μM, each of dGTP and dATP. Reactions also contained the indicated amounts of RFC, PCNA, and Pol α and were pre-incubated for 5 min at 30°C. DNA synthesis was initiated by adding 15 μl of 60 μM dCTP, 20 μM dTTP, 15 μCi of . Proteins were brought to a final volume of 200 μl in 100 mM sodium phosphate, 150 mM NaCl pH 8.0, and incubated with 50 μl (as a 10% slurry) Strep-Tactin magnetic beads  for 1 hr at 4°C with end-over-end mixing. Beads were collected in a magnetic separator and washed twice in 200 μl 100 mM sodium phosphate, 150 mM NaCl pH 8.0, then eluted in 75 μl 10 mM biotin in 100 mM sodium phosphate, 150 mM NaCl pH 8.0 for 20 min on ice. Samples were analyzed in 8% SDS-PAGE followed by staining with Coomassie Blue Denville stain.


AbstractSymmerista Hübner  is reviewed for Costa Rica, based on 49 wild-caught specimens. Four species are newly described: Symmerista luisdiegogomezi Chacón, Symmerista inbioi Chacón, Symmerista minaei Chacón and Symmerista aura Chacón. All are from the cloud forests of the Talamanca moutain range, southern Costa Rica. Photographs of the adults, male and female genitalia, and barcodes are also provided. The species Symmerista tlotzin Schaus (1892) is removed from Symmerista and assigned to the genus Elymiotis Walker as a new combination.The genus  Notodontidae includes about 3500 described species worldwide , from the cloud forests at altitudes between 1000 and 2600 meters.Hübner (1821) established the genus  species . SpeciesSymmerista tlotzin  male genitalia morphology and barcodes , Santo Domingo de Heredia, Costa Rica were examined, sexed and identified. Of those, 12 were DNA barcoded and dissected. All holotypes and paratypes are deposited in the collections at INBio. Fifty-six specimens of reared and wild-caught Elymiotis tlotzin, previously known as Symmerista tlotzin, were also examined.Forty-nine specimens of  Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa RicaINBio National Museum of Natural History, Smithsonian Institution, Washington DC, USAUSNM Adterminal lineAD Corpus bursaeCB Ductus bursaeDB ForewingFW HindwingHW Medial lineM Postmedial linePM Sternum 8ST8 Tergum 8T8 Wing lengthWLPageBreakTaxon classificationAnimaliaLepidopteraNotodontidaeHübnerSymmerista Hübner, 1821. Verz. bekannt. Schmett. : 248.Noctua albicosta Hübner, 1809, Samml. eur. Schmett. 4: pl. 93, fig. 440. By subsequent designation by, Kirby, 1892, Syn. Cat. Lepid. Het. Het. 1: 572.Noctua albicosta occurs in Canada and the US.Type(s), [North America]: Mistakenly included by Hübner as a European species; Symmerista was originally proposed in the Noctuidae.Nye (1975) stated: Noctua albicosta is a form of Phalaena albifrons Smith, 1797, in Smith & Abbot, Nat. Hist. rarer lepid. Insects Georgia 2: 159, pl. 80.Watson et al. (1980) stated: Adults – Medium-sized notodontid moth, FW = 16–22 mm, females larger than males; male antenna pectinate nearly to apex, terminal 10–12 annulations simple or antenna bipectinate, six terminal flagellomeres without rami; antennae of female simple; labial palpus porrect; haustellum reduced to two small lobes, completely hidden by labial palpi; ocelli absent; eye smooth, round; thorax generally dark brown with beige tegula; all scales of thorax long and forked; scales of patagium and prothorax beige and cream colored. FW with the costa straight, apex marked, the outer margin evenly rounded; accessory cell present, narrow; R2-5, stalked, R5 arising beyond R2, M1 from apex of accessory cell or nearly so. Male genitalia – ST8 with a wide, deep emargination; valva membranous, finely pubescent, costulae absent; tegumen narrowed dorsally; uncus concave, the socii subquadrate and lobed or long, wide, pubescent at bases, narrow and flattened at apices; vinculum slightly sclerotized; manica membranous, fused to juxta; ventral process of phallus long and forked, its tip well beyond the tip of the medial, dorsal process; vesica bulbous with a scobinate patch.Female genitalia – Papillae anales membranous, covered with short, scattered setae; posterior apophyses long and slender; DB sclerotized for approximately two-thirds of its length; CB rounded; anterior vaginal plate asymmetrical, slightly swollen and inflated in middle.Symmerista is characterized by: haustellum reduced to two small lobes, completely hidden by labial palpi; valva membranous; costulae absent; uncus concave; manica membranous, fused to juxta and ductus bursae sclerotized for approximately two-thirds of its length. Symmerista differs of the genera Elasmia and Elymiotis by the following characteristics: Elasmia has a well-developed haustellum, sacculus pleated and saccular scent organ present, manica sclerotized, deciduous cornuti, costulae present, costal margin sclerotized, ST8 with long and curvy lateral sclerotized projections like forceps, midplate with tiny pleats and membranous; uncus elongate, setose, with two dorsal protuberances and two tiny spines at the apex. Elymiotis has the haustellum well developed, sacculus pleated, deciduous cornuti, costal margin sclerotized, St8 posterior margin irregularly sclerotized and convex with tiny lateral proyections, uncus thin and long with apex acute and setose. All of these characterisctics are absent in Symmerista.The genus PageBreakTaxon classificationAnimaliaLepidopteraNotodontidaeChacónsp. n.http://zoobank.org/81A8E740-7368-45A7-9BBE-6113BBC43D3E16 specimens Holotype male: INB0003536238 , Costa Rica, Prov. Cartago, P.N. Tapanti, Macizo de La Muerte, Est. La Esperanza 9.69129-83.87683, 2600 m, September 2002, R. Delgado (INBio).Paratypes: 13 males, 2 females. 2 males: INB0003756237, INB0003756364 Costa Rica, Prov. Limon, Parque Internacional La Amistad, Valle del Silencio, Alrededor del Refugio y el Sendero Circular, 9.110281-82.961934, 2450 m, 22–27 September 2003, D. Rubi, R. Gonzalez, R. Delgado (INBio). 4 males: INB0003316532, INB0003316533, INB0003316534, INB0003316535 Costa Rica, Prov. Cartago, El Guarco, Macizo de la Muerte, Sector La Esperanza, 9.686771-83.87775, 2600 m, June 2001, R. Delgado (INBio). Male: INB0003352709 Costa Rica, Prov. Cartago, Reserva Forestal Rio Macho. El Guarco, Macizo de la Muerte, Sector La Esperanza, 9.686771-83.87775, 2600 m, August 2001, R. Delgado (INBio). 2 males: INB0003387641 (dissected), INB0003387642, Costa Rica, Prov. Cartago, Reserva Forestal Rio Macho, El Guarco, Macizo de la Muerte, 9.686771-83.87775, 2600 m, October 2001, R. Delgado (INBio). 2 males: INB0003536234 (COI barcoded), INB0003536235 (COI Barcoded), INB0003536237 (COI barcoded), Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de La Muerte, Est. La Esperanza, 9.69129-83.876832, 2600 m, September 2002, R. Delgado (INBio). 2 males: INB0003545221 , INB0003545222 (COI barcoded) Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de La Muerte, Est. La Esperanza, 9.69129-83.876832, 2600 m, October 2002, R. Delgado (INBio). Female: INB0003339209  Costa Rica, Prov. Cartago, El Guarco, Macizo de la Muerte, Est. La Esperanza. 9.686771-83.87775, 2600 m. July 2001. R. Delgado (INBio). Female: INB0003756274 Costa Rica, Prov. Limon, Parque Internacional La Amistad, Valle del Silencio, Alrededor Refugio y Sendero Circular, 9.110281-82.961934, 2450 m, 22–27 September 2003, D. Rubi, R. Gonzalez, R. Delgado (INBio).This species is named in honor of the late Professor Luis Diego Gómez Pignataro of San Jose, Costa Rica, for his outstanding contribution to our knowledge of Costa Rican biodiversity, his support of the Museo Nacional de Costa Rica, and for inspiring me to become a naturalist and taxonomist.Male genitalia: St8 posterior margin concave with a pair of short postero-lateral projections, projections robust and heavily sclerotized with blunt apices; valva membranous, finely pubescent, sacculus smooth, its margin uniform, costa straight with a distal protuberance near apex; valva with two triangular spine-like processes, one at base, the other at juxta near anellus; uncus slightly concave, somewhat helmet shaped, dorsal surface PageBreakrough, pubescent, ventral surface smooth with sparse pubescence; socii long, wide, pubescent at bases, narrow and flattened at apices, shape as in the figure; length of the phallus 3.1 mm, proximal part of phallus tube wide at the base, narrow in the middle, distal part of phallus tube robust, sclerotized, with a tubular lateral projection and rounded apex; proximal part of the vesica with a ventral scobinate patch, distal part of the vesica bulbous. Female genitalia: posterior apophyses longer and more slender than anterior apophyses; CB rounded and membranous, lacking a signum; DB wide and sclerotized; posterior margin of postvaginal plate sclerotized and emarginate.Dorsal FW ground color dark brown with costal margin black; an irregular, long thin cream-colored mark from the reniform spot to the apex; fringe dark brown with beige scales where veins touch termen; dorsal HW dark brown. Male , southern Costa Rica .Holotype male: INB0003487050 , Costa Rica, Prov. Cartago, El Guarco, Reserva Forestal Rio Macho, Macizo de la Muerte, Sector La Esperanza 9.686771-83.87775, 2600 m, May 2002, R. Delgado (INBio).Paratypes: Male: INB0003153991 Costa Rica, Prov. Cartago, El Guarco, San Isidro, Est. La Esperanza 9.687685-83.884582, 2450 m, March 2001, R. Delgado (INBio). 2 males: INB0003316531, INB0003316537 Costa Rica, Prov. Cartago, El Guarco, Macizo de la Muerte, Sector La Esperanza, 9.686771-83.87775, 2600 m, June 2001, R. Delgado (INBio). Male: INB0003320716 Costa Rica, Prov. Cartago, Parque Nacional Tapanti, El Guarco, San Isidro, Est. La Esperanza, 9.683922-83.876688, 2600 m, May 2001, R. Delgado (INBio). Male: INB0003334468 Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de la Muerte, Est. La Esperanza, 9.686771-83.87775, 2600-2700 m, April 2001, R. Delgado (INBio). 3 males: INB0003339195, INB0003339197, INB0003339198 Costa Rica, Prov. Cartago, El Guarco, Parque Nacional Tapanti, Macizo de la Muerte, Est. La Esperanza 9.686771-83.87775, 2600 m, July 2001, R. Delgado (INBio). Male: INB0003387640 Costa Rica, Prov. Cartago, Reserva Forestal Rio Macho, El Guarco, Macizo de la Muerte, 9.686771-83.87775, 2600 m, October 2001, R. Delgado (INBio). Male: INB0003478225 , Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de La Muerte, Est. La Esperanza, 9.69397-83.854504, 2700 m, 13–14 May 2002, J. Montero (INBio). 2 males: INB0003536236 , INB0003536239 (COI Barcoded) Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de La Muerte, Est. La Esperanza, 9.69129-83.876832, 2600 m, September 2002, R. Delgado (INBio). Male: INB0003545219  Costa Rica, Prov. Cartago, Parque Nacional Tapanti, Macizo de La Muerte, Est. La Esperanza, 9.69129-83.876832, 2600 m, October 2002, R. Delgado (INBio). 3 males: INB0003756229, INB0003756321, INB0003756418 Costa Rica, Prov. Limon, Parque Internacional La Amistad, Valle del Silencio, Alrededor del Refugio y Sendero Circular, 9.110281-82.961934, 2450 m, 22–27 September 2003, D. Rubi, R. Gonzalez, R. Delgado(INBio). Male: INBIOCRI001359567 Costa Rica, Prov. Cartago, Quebrada Segunda, Parque Nacional Tapanti, 9.762583-83.788328, 1250 m, February 1993, G. Mora. Male: INBIOCRI002210601 Costa Rica, Prov. Cartago, La Represa, Tapanti, 9.695643-83.768399, 1800 m, July 1995, R. Delgado (INBio). Male: INBIOCRI002253344 Costa Rica, Prov. Cartago, Rio Grande de Orosi, desde Puente Rio Dos Amigos hasta la Represa, 9.695643-83.768399, 1400-1800 m, March 1995, R. Delgado (INBio). Male: INBIOCRI002423774 Costa Rica, Prov. Cartago, Rio Grande de Orosi, desde Puente Rio Dos Amigos hasta la Represa, 9.695643-83.768399, 1800 m, February 1995, R. Delgado (INBio). Male, INBIOCRI002427627 Costa Rica, Prov. Cartago, Rio Grande de Orosi, desde Puente Rio Dos Amigos hasta la Represa, 9.695643-83.768399, 1400-1800 m, 22 August–15 September 1995, R. Delgado (INBio).PageBreakThis species is dedicated to the Instituto Nacional de Biodiversidad (INBio) in recognition of its 25 years of support for developing an understanding of the biodiversity of Costa Rica and exporting that understanding to the nation and the world.Symmerista inbioi differs from Symmerista luisdiegogomezi on: dorsal FW ground color light brown; from the reniform spot to the apex there is an uniform long, thin, white cream-colored band; fringe reddish brown; dorsal HW dirty beige. Male genitalia: St8 wide at base, anterior margin convex, posterior margin slightly irregular with a pair of very short projections, these sclerotized with blunt apices; valva membranous, saccular margin slightly jagged, costal margin smooth, with a distal protuberance near the apex; elongate finger-shaped process on the saccular margin at the internal base of the valve; uncus plate slightly concave, with papillae and setae on the dorsal and ventral edge; socii elongated, broad at base, narrow and flattened at the apex, with papillae and setae in the dorsal surface; length of the phallus 3.9 mm, proximal part of phallus curved at the base, slightly narrow in the middle, distal part of phallus tube sclerotized, narrow at the base, robust, irregular and wide at the end, with a tubular lateral projection with the distal nipple; proximal part of the vesica with a dorsal scobinate patch, distal part of the vesica bulbous. The genital armature of Symmerista inbioi is more robust and larger than Symmerista luisdiegogomezi.Male  (n Range) .DNA barcode of holotype male INB0003487050Symmerista inbioi | COI-5P:MHMXP003-08 | INB0003487050 | AACATTATATTTTATTTTTGGGATTTGAGCAGGTATAGTAGGAACTTCTTTAAGTCTATTAATTCGAGCTGAATTAGGAAACCCCGGATCACTTATTGGGGATGATCAAATTTATAATACAATTGTTACAGCCCATGCCTTTATTATAATTTTTTTTATGGTAATACCTATTATAATTGGGGGATTTGGTAATTGATTAGTCCCTCTTATACTAGGAGCCCCAGATATAGCATTCCCCCGCATAAATAATATAAGTTTTTGACTTTTGCCCCCTTCTTTAACCCTTTTAATTTCAAGAAGAATCGTAGAAAATGGAGCAGGAACTGGATGGACAGTGTACCCCCCACTATCCGCCAACATTGCCCATAGTGGAAGTTCTGTAGATTTAGCTATTTTTTCCCTTCATTTAGCTGGAATTTCCTCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAATATACGCCTCAATAATATATCTTTTGATCAAATACCTTTATTTGTTTGAGCTGTTGGAATTACAGCATTTTTACTTTTACTTTCTTTACCTGTTTTAGCGGGAGCTATTACAATACTACTAACTGACCGTAATTTAAATACATCCTTTTTTGACCCTGCTGGGGGAGGAGATCCAATTTTATACCAACATTTATTTTaxon classificationAnimaliaLepidopteraNotodontidaeChacónsp. n.http://zoobank.org/71A2F18E-A8EC-4BF6-9991-ACEC39D207994 specimens Holotype female: INB0003339208 , Costa Rica, Prov. Cartago, El Guarco, Macizo de la Muerte, Estacion La Esperanza, 9.68677-83.87775, 2600 m, July 2001, R. Delgado (INBio). Paratypes: Female: INB0003155283 (COI barcoded), Costa Rica, Prov. Limon, Bratsi, Valle del Silencio, 9.107197-82.961749, 2472 m, 11–12 October 2000, R. Delgado (INBio). Female: INB0003352700 (COI barcoded), Costa Rica, Prov. Cartago, Reserva Forestal Rio Macho, El Guarco, Macizo de la Muerte, Sector La Esperanza, 9.686771-83.87775, 2600 m, August 2001, R. Delgado (INBio).Other material examined: 1 Male, INB00033387642 (dissected) Costa Rica, Prov. Cartago, Reserva Forestal Rio Macho, El Guarco, Macizo de la Muerte, 9.68677-83.87775, 2600 m, October 2001. R. Delgado (INBio).This species is dedicated to the Ministerio del Ambiente y Energía (MINAE) of the government of Costa Rica in recognition of its 28 years of continuous and widespread support for the survival and conservation of the wild biodiversity of Costa Rica.PageBreakSymmerista minaei differs from Symmerista luisdiegogomezi on: dorsal FW ground color beige and light brown, square mark creamy near the reniform spot; beige mark in the apex; fringe beige yellow; dorsal HW beige. Male genitalia: T8 anterior margin slightly concave, posterior margin finely serrated with a window in the center; St8 lateral margins wide at the base, narrow to posterior margin, anterior margin concave, slightly sclerotized with a short projection in the center, posterior margin with robust projections, highly sclerotized on each side, with blunt apices, a little dome in the middle of the posterior margin; length of the phallus 3.3 mm, proximal part of phallus tube wide at base, distal part of phallus tube robust, sclerotized, with a tubular lateral projection rounded apex with the distal nipple; proximal part of the vesica with a ventral scobinate patch, distal part of the vesica bulbous. Female genitalia: Anterior and posterior apophyses the same size, long an slender; DB sclerotized; CB rounded, membranous and pleated; posterior margin of postvaginal plate sclerotized, slightly irregular, inverted V-shape.Male  (n Range) .DNA barcode paratype female INB0003155283.Symmerista minaei | COI-5P:MHMXP006-08 | INB0003155283 | PageBreakTATTATAATTTTTTTTATAGTAATACCTATTATAATTGGGGGATTTGGTAATTGATTAGTCCCCCTTATGCTAGGAGCCCCAGATATAGCATTCCCACGTATAAATAATATAAGTTTTTGACTTTTACCCCCCTCCTTAACCCTTTTAATTTCAAGAAGAATCGTCGAAAATGGGGCAGGAACCGGATGGACAGTGTACCCCCCACTATCCTCCAATATTGCCCACAGTGGAAGTTCTGTAGATTTAGCTATTTTTTCCCTACATTTAGCTGGAATTTCATCAATTTTAGGGGCCATTAATTTTATCACAACAATTATTAATATACGTCTCAATAACATATCTTTTGATCAAATACCCTTATTTGTTTGAGCTGTTGGAATTACAGCATTTTTACTTTTACTTTCTTTACCTGTTCTAGGGAGCTATTACAATACTACTAACGGATCGTAATTTAAATACATCTTTTTTTGATCCTGCAGGAGGAGGAGATCCAATTTTATATCAACATTTATTTAACATTATATTTCATTTTTGGAATTTGAGCAGGTATAGTTGGAACTTCATAAGCCTATTAATTCGAGCTGAATTAGGAAATCCCGGATCCCTTATTGGAGATGATCAAATTTATAACACAATTGTTACAGCCCATGCCTTTaxon classificationAnimaliaLepidopteraNotodontidaeChacónsp. n.http://zoobank.org/31DCFCFA-B792-406E-A667-CAAA17B5111F4 specimens Holotype male: INB0003116415 , Costa Rica, Prov. Cartago, Paraiso, P.N. Tapanti, Macizo de La Muerte, Estacion Quebrada Segunda, 9.762583-83.788328, 1300 m, November 2000, R. Delgado (INBio).Paratypes: 1 males, 2 females. Male: INBIOCRI002442681 (COI barcoded), Costa Rica, Prov. Puntarenas, Buenos Aires, Potrero Grande, Estacion Altamira, 1 Km. S del Cerro Biolley, 9.032987-83.010887, 1450 m, 13–26 May 1996, R. Villalobos (INBio). Female: INBIOCRI002549508 (COI barcoded), Costa Rica, Prov. Puntarenas, Coto Brus, Sabalito, Send. El ripario a 3 Km NE. de Progreso, 8.917676-82.78469, 1300 m, 6–9 April 1997, A. Picado (INBio). Female: INBIOCRI002754030 Costa Rica, Prov. Cartago, Turrialba, Tayutic, Moravia de Chirripo Shipiri, 9.837781-83.453639, 1000 m, 10 May 1983, D. H. Janzen & W. Hallwachs (INBio).This species is dedicated to Isidro Chacón’s daughter, Aura Chacón, for 25 years of understanding an absent father obsessed with his work.Male genitalia: St8 anterior margin with a sclerotized triangular projection in the middle, posterior margin sclerotized, irregular, with a rectangular projection at the center bearing two highly sclerotic and sharply serrated structures; proximal part of phallus tube wide and short at the base, with a blade-like lateral projection, sharp at the distal apex; a small rounded projection and curve off the previous, distal part of phallus tube robust, sclerotized, with a small bulge on the ventral side; proximal part of the vesica with a ventral scobinate patch, distal part of the vesica bulbous. Female genitalia: papillae anales ovoid-triangular, setose; posterior apophyses longer and more slender than anterior apophyses; DB sclerotized; CB elongated, membranous and pleated, lacking a signum; margin distal of antevaginalis plate very sclerotized and lightly depressed at the center, concave to the sides, proximal margin convex.Dorsal FW ground color light gray; a white cream mark from the discal cell to the apex. PageBreakSymmerista aura differs from female of Symmerista meridionalis Thiaucourt, 2007 in the shape of the antivaginalis plate; the CB elongated, membranous and pleated, lacking a signum; margin distal of antevaginalis plate very sclerotized and lightly depressed at the center, concave to the sides, proximal margin convex. This is the description of the female genitalia of Symmerista meridionalis published by Thiaucourt in 2007 which mentiones the differences, “Female terminalia: distal edge of lamella postvaginalis slightly incurved; lamella antevaginalis rectangular, almost square; its distal margin strongly sclerotized; mouth of the ostium bursae oval, densely sclerotized under the margin; ductus bursae beyond the constriction under the ostium, forming a short funnel; bursa membranous inserted on the dorsal surface of the extremity of the duct; signum distinct.” The male of Symmerista meridionalis is unknown.The female of Male   .DNA barcode paratype male INB0003116415.Symmerista aura | COI-5P:MHMXP017-08 | INB0003116415 | ACATTATATTTTATTTTTGGGGTTTGAGCTGGGATAGTTGGAACTTCCCTAAGTTTACTAATTCGAGCTGAATTGGGTAACCCTGGATCTTTAATTGGAGATGATCAAATTTATAATACAATTGTAACAGCCCATGCTTTTATTATAATTTTTTTTATAGTTATACCCACCATAATCGGGGGATTTGGTAATTGACTAGTTCCTCTTATATTAGGGGCACCGGATATAGCATTTCCACGTATAAATAACATAAGTTTTTGACTTCTACCCCCTTCTTTAACCCTTTTAATTTCAAGAAGAATTGTCGAAAATGGAGCTGGAACAGGATGAACAGTGTACCCCCCATTGTCATCTAATATTGCTCATGGTGGTAGTTCCGTAGATTTAGCTATTTTTTCACTTCATTTAGTGGAATTTCTTCAATTTTAGGGGCTATTAATTTTATTACAACAATCATTAATATACGTCTTAATAATATATCTTTTGACCAAATACCTTTATTTGTGTGAGCTGTAGGGATTACAGCATTTTTACTTTTACTTTCTTTACCTGTATTAGCTGGAGCTATTACAATATTATTAACTGATCGTAATCTAAACACATCTTTTTTTGATCCCGCTGGAGGAGGAGATCCTATTTTATACPageBreakTaxon classificationAnimaliaLepidopteraNotodontidaecomb. n.11 males 12 females.PageBreakINB0003319696 Costa Rica, Prov. Guanacaste, Nicoya, P.N. Barra Honda, Sector Barra Honda, 10.169826-85.379137, 50 m, 25–30 December 2000, H. Mendez (INBio). Female: INBIOCRI001184551 Costa Rica, Prov. Guanacaste, Bagaces, P. N. Palo Verde, Estacion Palo Verde, 10.349119-85.352345, 10 m, 20 June 1993, U. Chavarria (INBio). Female: INB0003300310 Costa Rica, Prov. Guanacaste, Hojancha, Z.P. Nosara, Hojancha, R.F. Monte Alto, 10.011248-85.402778, 400 a 500 m, 27 July – 3 August 2000, H. Mendez (INBio). Female: INBIOCRI000674401 Costa Rica, Prov. Guanacaste, Liberia, P.N.Sta. Rosa, Playa Naranjo, 10.802713 – 85.67479, 0–10 m, March 1991, E. Alcazar (INBio). Female: INB0003956448 Costa Rica, Prov. Guanacaste, Nicoya, San Antonio, Humedal Mata Redonda, 10.328094-85.42111, 8 m, 6 July 2005, B. Gamboa, J. Azofeifa, J. Gutierrez, M. Moraga, Y. Cardenas (INBio).2 Males: INBIOCRI000584965 Costa Rica, Prov. Guanacaste, Bagaces, Ref. Nac. Fauna Silv. R. L. Rodriguez, Estacion Palo Verde, 10.349119-85.352345, 10 m, May 1991, U. Chavarria (INBio). Male: INB0003072431 Costa Rica, Prov. Guanacaste, Bagaces, Pque. Nal. Palo Verde, Sector Palo Verde, 10.366668-85.383266, 0–50 m, 3 May 2000, H. Mendez (INBio). Male: INBIOCRI000386810 Costa Rica, Prov. Guanacaste, Liberia, P. N. Sta. Rosa, Playa Naranjo, 10.80275-85.666572, 0–10 m, May 1991, E. Alcazar (INBio). Male: INBIOCRI002426620 Costa Rica, Prov. Guanacaste, Liberia, Sector Las Pailas, 4.5 Km. SW del Volcan Rincon de la Vieja, 10.776784-85.351913, 800 m, 24 June 1995, K. Taylor (INBio). Male: INB0004065577 Costa Rica, Prov. Guanacaste, Liberia, Santa Rosa Nat. Pk., 10.83641-85.615491, 300 m, 4–6 December 1979, D. H. Janzen (INBio). Male: Male: 94-SRNP-2964 Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Estero Naranjo, 10.80426-85.68285, 2 m, 9 June 1994, Gusaneros. Male: 98-SRNP-9137 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Area Administrativa, 10.83764-85.61871, 295 m, 14 August 1998, Manuel Pereira. Male: 01-SRNP-17295 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero Carbonal, 10.77594-85.65799, 7 m, 2 November 2001, Gusaneros. Male: 06-SRNP-13290 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Estero Naranjo, 10.80426-85.68285, 2 m, 31 May 2006, Eilyn Camacho.PageBreakGuanacaste, Prov. Guanacaste, Sector Santa Rosa, Area Administrativa (adult at light), 10.83764-85.61871, 295 m, 1 June 2011, Daniel H. JanzenFemale: 92-SRNP-736 Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Vado Nisperal, 10.80212-85.65372, 10 m, 20 May 1992, Gusaneros. Female: 96-SRNP-1331 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero Palo Seco, 10.79342-85.6666, 5 m, 31 May 1996, Gusaneros. Female: 96-SRNP-1332 Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero Palo Seco, 10.79342-85.6666, 5 m, 2 June 1996, Gusaneros. Female: 98-SRNP-9134 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Area Administrativa (adult at light), 10.83764-85.61871, 295 m, 2 August 1998, Guillermo Pereira. Female: 98-SRNP-9137 Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Area Administrativa (adult at light), 10.83764-85.61871, 295 m, 14 August 1998, Guillermo Pereira. Female: 01-SRNP-17336 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero Carbonal, 10.77594-85.65799, 7 m, 28 October 2001, Gusaneros. Female: 01-SRNP-17336 Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero Carbonal, 10.77594-85.65799, 7 m, 28 October 2001, Gusaneros. Female: 07-SRNP-112736 (COI barcoded), Costa Rica, Area Conservacion Guanacaste, Prov. Guanacaste, Sector Santa Rosa, Sendero los Patos (adult at light), 10.82097-85.63323, 251 m, 8 December 2007, H. Cambronero & S. Rios. Female: 11-SRNP-12732 (COI Barcoded), Costa Rica, Area Conservacion PageBreakAdults –  in this genus, the species should be now in another genus.” KIRBY (1892) lists Edema  as a junior synonym of Symmerista Hubner, [1821].Nystaleini, but it differs from that of Symmerista .Eulophidae:Euplectrus (n=1).Elymiotis tlotzin have been collected in the dry forest ecosystem of Peninsula de Nicoya, and in the dry forests of Sector Santa Rosa and Sector Pailas of ACG, at elevations of 0 to 800 m. (Adult o 800 m. .DNA barcode female 11-SRNP-12732.Elymiotis tlotzin | COI-5P:MHMYM2073-11 | 11-SRNP-12732 | PageBreakAACATTATATTTTATTTTTGGAATTTGAGCAGGAATAGTAGGAACTTCTTTAAGTTTATTAATTCGAGCTGAATTAGGAAATCCAGGATCTTTAATTGGTGATGATCAAATTTATAATACTATTGTAACAGCTCATGCTTTTATTATAATTTTTTTTATAGTAATGCCTATTATAATTGGAGGATTTGGAAATTGACTAGTTCCATTAATATTAGGAGCCCCAGATATAGCTTTCCCCCGAATAAATAATATAAGATTTTGACTACTTCCACCCTCACTAACTTTATTGATTTCAAGAAGTATTGTAGAAAATGGAGCAGGAACTGGATGAACAGTTTATCCCCCCCTTTCATCTAA0TATTGCACATAGAGGAAGATCTGTAGATTTAGCAATTTTTTCACTTCATTTAGCTGGTATTTCATCGATTTTAGGAGCTATTAATTTTATTACAACGATTATTAATATACGACTTAATAACATAACTTTTGATCAAATACCTTTATTTGTTTGAGCAGTAGGAATTACAGCTTTTTTATTATTATTATCTTTACCTGTTTTAGCCGGAGCGATTACTATATTATTAACAGACCGTAATTTAAATACTTCATTTTTCGACCCTGCTGGTGGAGGAGATCCAATTCTTTATCAACATTTATTT


Similar results were obtained using cell size (forward/side scatter) to fractionate MCF7 cells. Larger stem-like cells also showed increased hTERT-GFP levels, as well as increased mitochondrial mass and function. Thus, this simple and rapid approach for the enrichment of immortal anabolic stem-like cancer cells will allow us and others to develop new prognostic biomarkers and novel anti-cancer therapies, by specifically and selectively targeting this metabolic sub-population of aggressive cancer cells. Based on our proteomics and functional analysis, FDA-approved inhibitors of protein synthesis and/or mitochondrial biogenesis, may represent novel treatment options for targeting these anabolic stem-like cancer cells.Tumor cell metabolic heterogeneity is thought to contribute to tumor recurrence, distant metastasis and chemo-resistance in cancer patients, driving poor clinical outcome. To better understand tumor metabolic heterogeneity, here we used the MCF7 breast cancer line as a model system to metabolically fractionate a cancer cell population. First, MCF7 cells were stably transfected with an hTERT-promoter construct driving GFP expression, as a surrogate marker of telomerase transcriptional activity. To enrich for immortal stem-like cancer cells, MCF7 cells expressing the highest levels of GFP (top 5%) were then isolated by FACS analysis. Notably, hTERT-GFP(+) MCF7 cells were significantly more efficient at forming mammospheres  and showed increased mitochondrial mass and mitochondrial functional activity, all relative to hTERT-GFP(−) cells. Unbiased proteomics analysis of hTERT-GFP(+) MCF7 cells directly demonstrated the over-expression of 33 key mitochondrial proteins, 17 glycolytic enzymes, 34 ribosome-related proteins and 17 EMT markers, consistent with an anabolic cancer stem-like phenotype. Interestingly, MT-CO2  expression was increased by >20-fold. As MT-CO2 is encoded by mt-DNA, this finding is indicative of increased mitochondrial biogenesis in hTERT-GFP(+) MCF7 cells. Importantly, most of these candidate biomarkers were transcriptionally over-expressed in human breast cancer epithelial cells  Telomerase plays a central role both in the biology of normal aging, as well as in the development of human cancers , 2. HoweRecently, Clarke and colleagues have taken advantage of the properties of human telomerase (hTERT), to enrich for a population of osteosarcoma cells with stem-like properties . For thiHere, we have adapted this approach to the study of breast cancer stem-like cells, with a focus on proteomics analysis, biomarker discovery and cell metabolism. Importantly, we demonstrate that hTERT-high-activity breast cancer cells form mammospheres with higher efficiency, and show a proteomics profile consistent with an anabolic cancer stem cell phenotype. In support of this notion, we also show that hTERT-high-activity breast cancer cells have increased mitochondrial mass and activity, consistent with an increase in mitochondrial biogenesis.To enrich for a population of immortal CSCs, we exploited a sensitive eGFP reporter system for the fluorescent detection of high telomerase transcriptional activity. Briefly, MCF7 cells were transduced with a lentiviral vector driving eGFP protein expression, under the control of a 1.5-kB fragment of the hTERT promoter. This DNA construct also contains a puromycin-resistance cassette for antibiotic-resistance selection. Schematic diagrams illustrating this overall experimental strategy and the construction of the hTERT-promoter-vector are shown in Figures After selection with puromycin, MCF7-hTERT-eGFP cells were subjected to FACS analysis to visualize the broad distribution of eGFP expression, which serves as a surrogate marker of telomerase activity. Importantly, fewer than 1 in a 100 cells in MCF7-hTERT-eGFP cell monolayers visually showed high GFP fluorescence. In striking contrast, there was a dramatic enrichment of GFP(+) cells in MCF7 cell mammospheres (> 50 μm), each containing usually 1-to-2 GFP-high cells  and GFP-low (negative) groups. Then, five thousands cells from each group were seeded per well in 6-well low-attachment plates. These two groups were compared to the total unfractionated cell population.Remarkably, Figure In order to dissect the mechanism(s) by which high telomerase activity drives the survival and clonal expansion of CSCs, we next employed an unbiased label-free proteomics approach. The proteome of fractionated MCF7-hTERT-eGFP cells was directly determined, after FACS separation into GFP-high and GFP-low populations. For simplicity, we focused on the proteins that were over-expressed in GFP-high cells by > 1.5-fold. Our results are summarized in Tables Importantly, >30 mitochondrial-related proteins were over-expressed in GFP-high cells Tables . Most ofCSCs undergo an EMT, which facilitates cell migration, invasion and metastatic dissemination . Thus, wElevated protein synthesis is another important feature of the anabolic CSC phenotype . As predTaken together, hTERT-eGFP-high cells over-express >100 proteins related to an anabolic CSC phenotype.in vivo. For this purpose, we exploited a clinical data set of tumor samples from N = 28 breast cancer patients. These tumor samples were subjected to laser-capture micro-dissection, to separate epithelial cancer cells from adjacent tumor stroma [in vivo. Tables To determine the clinical relevance of our findings, we next assessed whether the hTERT proteomic targets that we identified in GFP-high cells were transcriptionally over-expressed, in human breast cancer cells r stroma . OverallTo directly validate the mitochondrial phenotype of hTERT-eGFP-high cells top 5%), we used two well-established fluorescent probes to quantitate mitochondrial membrane potential and mass, by FACS analysis. More specifically, we used MitoTracker Orange (561-nm) as a reporter for mitochondrial membrane potential and MitoTracker Deep-Red 640-nm) as a marker of mitochondrial mass. Importantly, Figures 0-nm as a%, we usein vivo [Previous studies using mouse mammary epithelial cells have demonstrated that stem-like cells can be enriched solely based on cell size . For exain vivo .larger cells  and ii) smaller cells   ) Figure . Interes) Figure .As such, larger cell size in MCF7 cells directly correlates with telomerase activity  and mitochondrial mass/activity, which would be consistent with an anabolic CSC phenotype. These results provide independent validation for the idea that high hTERT activity (“stemness”) is functionally associated with increased mitochondrial mass and activity in breast cancer cells, and co-segregates with large cell size. Importantly, large cell size is determined by increased PI3K/AKT/mTOR-signaling, which drives significant increases in overall protein synthesis –14. ThisHere, we have used an hTERT-promoter-eGFP-reporter system to identify and purify a sub-population of MCF7 cells, with high hTERT transcriptional activity, by FACS analysis. These hTERT-eGFP-high cells formed mammospheres with greater efficiency, as predicted, consistent with the idea that this sub-population of cells is enriched in cancer stem-like cells. Importantly, proteomics analysis of these hTERT-eGFP-high MCF7 cells revealed the upregulation of mitochondrial proteins, glycolytic enzymes and EMT markers, as well as components of the protein synthesis machinery, such as ribosome-related proteins and chaperones for protein folding. Interestingly, MT-CO2  expression was increased by >20-fold. As MT-CO2 is encoded by mt-DNA, this finding is indicative of increased mitochondrial biogenesis in hTERT-eGFP-high MCF7 cells. We then functionally validated that hTERT-eGFP-high MCF7 cells show increases in mitochondrial mass and activity, using two distinct MitoTracker probes. Complementary results were obtained using cell size to fractionate MCF7 cells. Larger stem-like cells showed increased hTERT-GFP levels, as well as increased mitochondrial mass and function. Thus, these two independent approaches for the enrichment of immortal anabolic CSCs should allow the development of new prognostic biomarkers and related novel anti-cancer therapies.Interestingly, recent studies in aging and cancer have both directly linked telomerase activity to mitochondrial function, via the hTERT-p53-PGC1 signaling axis –18. Moreprotein synthesis machinery  and ii) are associated with large cell size , which share several properties with CSCs or TICs –26. For We recently directly compared the proteome of MCF7 cell monolayers to MCF7-derived mammospheres, using label-free unbiased proteomics analysis. Consistent with our current findings, this analysis demonstrated that MCF7-derived mammospheres show the over-expression of >60 mitochondrial-related proteins and >80 components of the protein synthesis machinery , 29. ComImportantly, functional validation studies revealed that mammosphere formation could be efficiently blocked with well-established inhibitors of mitochondrial function and/or inhibitors of protein synthesis, such as oligomycin A and puromycin, respectively , 29. Morin vivo. Thus, the new hTERT metabolic targets that we identified here may be important for improving human breast cancer diagnosis and therapy.In summary, we show here that high telomerase activity metabolically defines a sub-population of anabolic CSCs, with increased mitochondrial mass and large cell size. Overall, greater than seventy hTERT targets  that we identified in hTERT-eGFP-high cells were also transcriptionally elevated in human breast cancer cells MCF7 cells were purchased from the ATCC. Gibco-brand cell culture media (DMEM and DMEM/F12) was purchased from Life Technologies. The lentiviral vector encoding the hTERT promoter linked to eGFP was custom-made by Genecopoeia (USA). MitoTracker probes (Deep Red and Orange) were purchased from Molecular Probes/Invitrogen, via Life Technologies. The telomerase inhibitor IX  was obtained commercially from Santa Cruz Biotech (USA).R cassette, and cloned into a lentiviral vector, custom-made by Genecopoeia: 5′-TAAAATTGTGTTTTCTATGTTGGCTTCTCTGCAGAGAACCAGTGTAAGCTACAACTTAACTTTTGTTGGAACAAATTTTCCAAACCGCCCCTTTGCCCTAGTGCAGAGACAATTCACAAACACAGCCCTTTAAAAAGGCTTAGGGATCACTAAGGGGATTTCTAGAAGAGCGACCTGTAATCCTAAGTATTTACAAGACGAGGCTAACCTCCAGGAGCGTGACAGCCCAGGGAGGGTGCGAGGCCTGTTCAAATGCTAGCTCCATAAATAAAGCAATTTCCTCC GGCAGTTTCTGAAAGTAGGAAAGGTTACATTTAAGGTTGCGTTTGTTAGCATTTCAGTGTTTGCCGACCTCAGCTACAGCATCCCTGCAAGGCCTCGGGAGACCCAGAAGTTTCTCGCCCCTTAGATCCAAACTTGAGC AACCCGGAGTCTGGATTCCTGGGAAGTCCTAGCTGTCCTGCGGTTGTGCCGGGGCCCCAGGTCTGGAGGGGACCAGTGGCCGTGTGGCTTCTACTGCTGGGCTGGAAGTCGGGCCTCCTAGCTCTGCAGTCCGAGGCTTGGAGCCAGGTGCCTGGACCCCGAGGTTGCCCTCCACCCTGTGCGGGCGGGATGTGACCAGATGTTGGCCTCATCTGCCAGACAGAGTGCCG GGGCCCAGGGTCAAGGCCGTTGTGGCTGGTGTGAGGCGCCCGGTGCGCGGCCAGCAGGAGCGCCTGGCTCCATTTCCCACCCTTTCTCGACGGGACCGCCCCGGTGGGTGATTAACAGATTTGGGGTGG TTTGCTCATGGTGGGGACCCCTCGCCGCCTGAGAACCTGCAAAGAGAAATGACGGGCCTGTGTCAAGGAGCCCAAGTCGCGGGGAAGTGTTGCAGGGAGGCACTCCGGGAGGTCCCGCGTGCCCGTCC AGGGAGCAATGCGTCCTCGGGTTCGTCCCCAGCCGCGTCTACGCGCCTCCGTCCTCCCCTTCACGTCCGGCATTCGTGGTGCCCGGAGCCCGACGCCCCGCGTCCGGACCTGGAGGCAGCCCTGGGTCTCCGGATCAGGCCAGCGGCCAAAGGGTCGCCGCACGCACCTGTTCCCAGGGCCTCCACATCATGGCCCCTCCCTCGGGTTACCCCACAGCCTAGGCCGATTCGACCTCTCTCCGCTGGGGCCCTCGCTGGCGTCCCTGCACCCTGGGAGCGCGAGCGGCGCGCGGGCGGGGAAGCGCGGCCCAGACCCCCGGGTCCGCCCGGAGCAGCTGCG CTGTCGGGGCCAGGCCGGGCTCCCAGTGGATTCGCGGGCACAGACGCCCAGGACCGCGCTTCCCACGTGGCGGAGGGACTGGGGACCCGGGCACCCGTCCTGCCCCTTCACCTTCCAGCTCCGCCTCCTCCGCGCGGACCCCGCCCCGTCCCGACCCCTCCCGGGTCCCCGGCCCAGCCCCCTCCGGGCCCTCCCAGCCCCTCCCCTTCCTTTCCGCGGCCCCGCCCTCTCCTCGCGGCGCGAGTT-3′.The following 1.5 kB sequence was used as the hTERT-promoter to generate the hTERT-eGFP-PuroR cassette were prepared and used to stably transduce MCF7 cells, according to the manufacturer's protocol (in the presence of 5 μg/ml polybrene). Twenty-four hours post-infection, media containing the virus was removed and replaced with standard media. Cells were then selected with puromycin (2 μg/ml), for up to 10 days. Please note that for most of the experiments described in this paper, we compared the GFP-high (top 5%) fraction to the GFP-low/negative (bottom 5%) fraction, unless stated otherwise. However, for label-free proteomics analysis, we compared the GFP-high (top 10%) population to the GFP-low (bottom 10%) population, to insure that enough material was collected for sample processing. For these experiments, singlet FACS gating of live cells was utilized.Lentiviral particles harboring the hTERT-eGFP-Puro2 in mammosphere medium (DMEM-F12/B27/20-ng/ml EGF/PenStrep) in non-adherent conditions, in culture dishes coated with (2-hydroxyethylmethacrylate) . Cells were grown for 5 days and maintained in a humidified incubator at 37°C at an atmospheric pressure in 5% (v/v) carbon dioxide/air. After 5 days for culture, spheres >50 μm were counted using an eye piece graticule, and the percentage of cells plated which formed spheres was calculated and is referred to as percentage mammosphere formation, and was normalized to one (1 = 100% MSE). Mammosphere assays were performed in triplicate and repeated three times independently.A single cell suspension of MCF7-hTERT-eGFP cells was prepared using enzymatic , and manual disaggregation (25 gauge needle) . Cells wCells were treated on day 0 with MST-312 at a concentration of 10 μM and compared to vehicle alone controls, processed in parallel, after 5 days of mammosphere culture. A dose-response analysis of the effects of MST-312 on parental MCF7 cells was first performed to establish an effective concentration for the inhibition of mammosphere formation; our results showed that 1 μM had little or no effect, while 10 μM inhibited mammosphere formation by approximately 70% (data not shown). Therefore, experiments with eGFP fractionated MCF7 cells were carried out at 10 μM.Fluorescent imaging of MCF7 cells in adherent conditions and non-adherent spheroid culture was performed using a Leica SP8 multi-photon DM6000 microscope to detect GFP expression and bright field images.Cell lysates were prepared for trypsin digestion by sequential reduction of disulphide bonds with TCEP and alkylation with MMTS . Then, tN = 28 human breast cancer patients) [To firmly establish the clinical relevance of our results from the quantitative proteomics, we re-analyzed the transcriptional profiles of epithelial breast cancer cells and adjacent tumor stromal cells that were physically separated by laser-capture microdissection (from atients) .For live cell sorting experiments, hTERT-eGFP transfected MCF7 cells were resuspended in PBS and sorted according to eGFP expression using the BD influx. Untransfected MCF7 cells were used as a negative control to determine positive GFP expression. Cells were sorted into two groups, those with the highest GFP expression and those with the lowest GFP expression. For FACs analysis experiments, hTERT-eGFP cells were labeled with MitoTracker Deep Red  and MitoTracker Orange . Cells were kept of ice until analysis using the FACS Calibur. Results were analyzed using FlowJo software version 10. Cells were gated into two populations, those with the highest 5% of GFP expression, and those with the lowest 5% and negative GFP expression. The median fluorescent intensity of MitoTracker Deep Red (640nm) and MitoTracker Orange (561nm) was then determined within the two distinct GFP cellular populations. Additionally, cells were discriminated by cell size into two populations, small and large using SSC (side scatter) and FSC (forward scatter). The median intensity of eGFP, MitoTracker Deep Red, and MitoTracker Orange, within these populations, was then determined.To measure mitochondrial activity, cells were stained with MitoTracker Orange , whose accumulation in mitochondria is dependent upon membrane potential. To measure mitochondrial mass, cells were stained with MitoTracker Deep Red , localizing to mitochondria regardless of mitochondrial membrane potential. Cells were incubated with pre-warmed MitoTracker staining solution  for 30-60 min at 37 °C. All subsequent steps were performed in the dark. Cells were washed in PBS, harvested, and re-suspended in 300 μL of PBS. Cells were then analyzed by flow cytometry. Data analysis was performed using FlowJo software.hTERT-eGFP-MCF7 cells, co-labeled with MitoTracker dyes, were analyzed by cell size using FlowJo software. Cells were separated by gating of forward scatter (FSC) and side scatter (SSC) plots into two populations for analysis, large cells  and small cells . The median fluorescent intensity of eGFP and MitoTracker dyes were determined in each population of cells. Very similar results were obtained, with either singlet FACS gating or all live cell FACS gating.Statistical significance was determined using the Student's t-test or ANOVA, where appropriate. Values of less than 0.05 were considered significant. Data are shown as the mean ± SEM, unless stated otherwise.


Drosophila Leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3), and find body asymmetries similar to those found in insulin-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNA intereference (RNAi) against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By tagging endogenous Lgr3 and performing cell type-specific RNAi, we map this Lgr3 activity to a new subset of CNS neurons, four of which are a pair of bilateral pars intercerebralis Lgr3-positive (PIL) neurons that respond specifically to ectopic Dilp8 by increasing cAMP-dependent signalling. Our work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations.How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. Here we mutate an invertebrate orphan relaxin receptor gene, the  The orphan ligand Dilp8 has been shown to coordinate growth and developmental timing in Drosophila. Here, using Gal4 drivers and CRISPR/Cas9 approaches, Garelli et al. identify a role for relaxin-like receptor Lgr3 in regulating the Dilp8 developmental delay pathway. Drosophila, the insulin/relaxin-like peptide (Dilp8), which ensures organ and body size coordination3dilp8 uncouples the endocrine communication between imaginal discs and the prothoracic gland, making dilp8 mutants susceptible to uncoordinated disc growth when intrinsic errors of development or noxious environmental stimuli affect the growth status of one or more discs. This results in an increase in random deviations from bilateral symmetry (fluctuating asymmetry (FA)), measurable at the population levelDrosophila3456How different organs in the body sense growth perturbations in distant tissues to coordinate their size and differentiation status during development is poorly understood1bona fide relaxin peptide homologues89Drosophila genome encodes two orphan receptors, Lgr3 and Lgr4, with clear homologies to vertebrate relaxin receptors  neurons.Type C1 Leucine-rich repeat-containing G protein-coupled receptors (Lgrs) are a conserved protein family in metazoans that act as receptors for insulin-like peptides of the relaxin subfamily in vertebrates, where they play diverse roles in tissue homeostasis and remodelling, behaviour and reproductionLgr3, we remobilized an MB Minos elementLgr3+/+), which served as genetic background controls, and one imprecise excision allele, Lgr3ag1, which consists of a 3.8-kb deletion that completely removes exon 8 and partially removes exon 9 (Lgr3ag1 produces a transcript with a premature termination codon (PTC) that truncates the Lgr3 protein one amino acid after D326  are increased by an order of ∼3 when compared with their Lgr3+/+ controls  under control of the wing-pouch Beadex-Gal4 (Bx>) driver3Lgr3+/+ animals ; To generate a mutant for s exon 9 . Lgr3ag1ter D326 . We concarea FAi ). This p animals . This deLgr3 should be necessary for the developmental delay produced by ectopic Dilp8 expression in the absence of tissue growth abnormalities3UASP-dilp8::3xFLAG (UAS-dilp8a) transgene under the control of armadillo-Gal4 (arm>) was suppressed in larvae homozygous for Lgr3ag1 or trans-heterozygous for Lgr3ag1 and a deficiency that completely uncovers the Lgr3 locus (Lgr3Df(3)BSC321) )tubulin-Gal4 (tub>), and reduced Lgr3 activity by concomitant RNA intereference (RNAi) knockdown using a short hairpin (TRiP VALIUM22 (Lgr3-IR-V22)) lineLgr3 mRNA levels by ∼85%  (Lgr3 completely suppressed the dilp8-dependent delay (Lgr3 (TRiP VALIUM10 (Lgr3-IR-V10))dilp8-dependent delay, albeit to a lesser extent than Lgr3-IR-V22 (Lgr3 mRNA levels (∼50%) than Lgr3-IR-V22  . Coherennt delay . A secon3-IR-V22 . This pa3-IR-V22 , suggestLgr3 at the protein level, we used CRISPR/Cas9-mediated homologous repair15Lgr3ag5, hereafter named sfGFP::Lgr3, which contained an intact Lgr3-coding sequence downstream of the sfGFP insertion  indels in the Lgr3-coding region , which induces apoptosis, tissue damage/regeneration, an imaginal-disc-specific Dilp8 upregulation and consequentially a robust delay in the onset of metamorphosissfGFP::Lgr3 ag5 allele encodes a functional receptor. Together, these results strongly suggest that the Lgr3 protein acts in a subpopulation of CNS neurons.To gain insight into the tissue and cellular expression pattern of nsertion . sfGFP::nsertion . No otheof Dilp8 . The effg region  or by usWe were unable to detect sfGFP::Lgr3 expression in neurons directly innervating the ring gland , suggestsfGFP::Lgr3 neurons in more detail. sfGFP::Lgr3 expression was not homogeneous in the ∼180 CNS cell bodies. It was most strongly expressed in a single pair of neurons in the very tip of the VNC, in a single dorso-ventral pair of midline neurons located deep in the thoracic segment of the VNC ), and in a pair of bilateral neurons localized in the anterior part of the pars intercerebralis ) -Gal4 driving a UAS-myr::tdTomato reporter indicates that many Lgr3-positive neurons are cholinergic, including all three major neuronal populations -Gal4 and Vesicular glutamate transporter promoter (VGlut)-Gal4, which drive expression in GABAergic and glutamatergic neurons, respectively, label very faintly the PIL neurons and the pair of distal VNC neurons and show no detectable staining in MIL neurons ) . All thrL1 stage . Co-stai neurons ). Co-sta neurons . MIL neu neurons . PIL neu neurons . The lat neurons . PIL neu neurons . These rLgr3-IR-V22 RNAi line to the panneuronal driver elav-Gal4 (elav>) and performed EMS assays -Gal4 did not rescue the EMS-induced delay  and GMR19B09-Gal4 (GMR19B09>)  throughout embryonic and larval development was compatible with developmental progression and led to a significant suppression of the EMS-dependent delay  , selecteMS-delay , the GMRhoc test . These rGMR19B09> driver labels ∼270 neurons, ∼30 of which populate each brain hemisphereGMR19B09> neurons with myr::tdTomato in a sfGFP::Lgr3 background, we detect overlapping expression in ∼10 neurons per hemisphere, 2 of which are the bright PIL neurons  line drives expression in similar type of neurons, named #5 neurons25. MZ699>myr::tdTomato expression analysis in a sfGFP::Lgr3 background demonstrates that PIL neurons are a subset of #5 neurons -(FRTmCherry) luciferase reporterCRE-luciferase and the sfGFP::Lgr3 reporters  in heterologous studies in human cell lines ery cell . We concDrosophila SL2/DL2 cells. In this assay, Dilp8 is covalently attached to TriCEPS2727Drosophila insulin-like receptor (InR)Drosophila cells  assays27la cells . An endoCTLH2) -like factor derailed (Drl), neuroglian (Nrg), the laminin receptor integrin α-PS3 (ITA3) and the choline transporter (CTL)-like protein 2 (CTLH2) . Any of CTLH2) . While tDrosophila. Our study opens many questions for further research, such as the determination of which of the eight bilateral Lgr3-positive interneuron populations , and relayed through one or more steps before reaching the Lgr3-positive cells , it is tempting to propose a direct ligand–receptor interaction between them. This possibility is supported by the strong genetic interaction between in vivo, nevertheless it strongly indicates that Dilp8 can consistently interact with a likewise strong receptor candidate for an Ilp, such as the InR. The LRC technique we used can identify receptors of interest with affinities spanning 4 orders of magnitude at expression levels as low as 2,000 receptors per cell . However, it is not yet clear how quantitative it can be relative to affinity constants. Dilp8 has been previously shown to modulate growth in vivo often in opposite ways depending on the observed tissueThor (4E-BP) in the larval fat body, which is consistent with a local increase in insulin/IGF-like signallingThor levels are higher in imaginal discs in the same animalsSecond, we have failed to identify Lgr3 among candidate Dilp8-binding cell surface receptors/co-receptors. Clearly, the biochemical identification of alternative cell surface-binding proteins such as the InR, Nrg and the RYK-like Drl37Third, the fact that ectopic expression of Dilp8 only leads to a detectable increase in cAMP signalling in PIL neurons, and not in other Lgr3-positive neurons , indicatsfGFP::Lgr3 or GMR19B09>myr::tdTomato expression in the ring gland or in neurons innervating the ring gland , UAS-rpr , Lgr3-IR-V10 , Lgr3-IR-V22 , elav-Gal4 (P{w[+mW.hs]=GawB}elavC155), elav-Gal4 (P{w[+mC]=GAL4-elav.L}2/CyO), w1118;Mi{ET1}Lgr3MB06848, w1118; snaSco/SM6a, P{w[+mC]=hsILMiT}2.4, tub-Gal4 , w1118; Lgr3Df(3)BSC321/TM6C Sb1 cu1, Gad1-Gal4 (P{w[+mC]=Gad1-GAL4.3.098}2/CyO), Cha-Gal4 , GMR19B09-Gal4 , GMR17G11-Gal4 , y1 w* P{y[+t7.7] w[+mC]=10XUAS-IVS-mCD8::RFP}attP18 P{y[+t7.7] w[+mC]=13XLexAop2-mCD8::GFP}su(Hw)attP8, and w1118; P{y[+t7.7] w[+mC]=GMR19B09-lexA}attP40w were obtained from the Bloomington Drosophila Stock Center at Indiana University. The stock 1 M{w[+mC]=Act5C-Cas9.P}ZH-2A w*y (reference #16) was obtained from Bestgene. The stock arm-gal4  was a gift from P. Domingos. The stocks UAS-dilp8b::3XFLAG and UAS-dilp8c::3XFLAG (reference #3) were a gift from M. Dominguez. The stock w*; P{GawB}Mz699 and UAS-myr::tdTomato/CyO; TM2/TM6B were a gift from M.L. Vasconcelos. Balanced lines were generated by crosses to the stock w1118; If/CyO; MKRS/TM6B, which was a gift from A. Jacinto. The stock w; CRE-F-luc (II) was a gift from J.C. Yin. The stock y w; ptth-HA was a gift from M. O'Connor. Stocks are maintained at low densities at 18 °C in a 12-h light/dark cycle.The Drosophila Lgr3 locus by using the MiET1 transposase to remobilize the MB Minos element Lgr3MB06848 (reference #11), which is inserted in the seventh Lgr3 intron, <100 bp from exon 7  and transferred to new vials every dayCy-Minos/+; Lgr3MB06848/MKRS or TM6B male adults were selected and individually crossed to the balancer strain w1118; If/CyO; MKRS/TM6B. A single eGFP-negative (lacking the Mi{ET1} element) male was selected from each cross and mated with w1118; If/CyO; MKRS/TM6B females. The putative Lgr3 excisions were balanced over TM6B to obtain the following genotypes w1118; +/+; Lgr3MB06848excision/TM6B. We obtained one imprecise excision that generated the Lgr3ag1 mutant allele and three precise excisions named Lgr3ag2, Lgr3ag3 and Lgr3ag4 all of which behaved the same way. In this study, the Lgr3ag2 line was used as the genetic background control for the imprecise excision allele Lgr3ag1.We generated a mutation in the m exon 7 . Virgin Lgr3ag1 deletion, we performed a series of PCR assays with primer pairs located around the Lgr3MB06848 insertion . This suggested that while the Lgr3ag1 deletion was very large, its breakpoints were confined within the Lgr3 locus. We then tried to amplify a 5.3-kb PCR product with a primer upstream of the Lgr3MB06848 position  are lacking in the Lgr3ag1 leucine-rich repeat domains, which are critical for relaxin ligand binding in vertebrate relaxin receptorsTo molecularly characterize the nsertion . Lgr3ag1r3 locus . This alLgr3ag1 and controls Lgr3+/+ and the original Lgr3MB06848 stock. Lgr3ag1 generated a smear with a major band that was ∼200 bp smaller than the other control genotypes –PCR analyses with mRNA isolated from o exon 9 . The resn 9 (326 . We concterminus .Drosophila and Homo sapiens codon-optimized complementary DNA (cDNA) corresponding to full-length Lgr3 was synthetized de novo and cloned into pUASPpUASP-Lgr3. This plasmid was injected into w1118 and two independent insertions were tested, pUASP-Lgr3a  and pUASP-Lgr3b . A similar protocol was used to place the dilp8::3xFLAG construct described in reference #3 into pUASP, making pUASP-dilp8a.A http://tools.flycrispr.molbio.wisc.edu/targetFinder/)We used a CRISPR/Cas9-mediated homologous repair strategy15gRNA 1Fw: 5′-CTTCGGAGCACTCAATTCCCACTC (CGG)-3′Rv: 5′-AAACGAGTGGGAATTGAGTGCTCC-3′gRNA 2Fw: 5′-CTTCGCAAACTCAAGTAGAATATCA (CGG)-3′Rv: 5′-AAACTGATATTCTACTTGAGTTTGC-3′The PAM regions (CGG) are located right after the forward primers of both gRNAs 1 and 2, as shown above.As a repair cassette we designed the following sequence, where each colour represents the following:Lgr3 ATG siteOrange: Homology region up to Lgr3 ATGRed: Green: sfGFPBlue: Spacer (GSGSGS)Lgr3 ATG siteViolet: Homology region after ATGCGTAAGGGCGAGGAGTTGTTCACGGGAGTTGTGCCCATATTGGTTGAGCTGGATGGAGATGTGAATGGCCACAAGTTCAGTGTGCGGGGTGAGGGAGAAGGAGACGCAACAAACGGTAAGCTGACACTGAAGTTCATTTGTACTACGGGCAAGCTCCCGGTGCCATGGCCCACATTGGTCACCACCCTGACCTATGGCGTGCAATGCTTCGCCCGATATCCAGATCATATGAAGCAGCATGATTTCTTTAAGTCGGCCATGCCCGAGGGTTACGTACAAGAGCGCACTATTAGCTTTAAGGACGACGGTACGTATAAAACCAGGGCTGAGGTGAAGTTTGAGGGTGATACCCTGGTGAACCGCATTGAATTGAAGGGCATCGATTTTAAGGAGGACGGCAACATCCTGGGCCACAAGCTCGAATATAATTTTAATAGCCATAATGTTTACATTACCGCGGACAAGCAGAAGAATGGAATTAAGGCTAATTTCAAGATCCGACATAATGTGGAGGACGGATCCGTTCAGTTGGCCGATCACTACCAGCAAAACACCCCCATCGGAGATGGCCCCGTCCTGCTGCCCGATAACCACTACCTGAGTACCCAGTCCGTCCTGTCGAAGGATCCTAATGAGAAGCGGGATCATATGGTGCTGCTGGAGTTTGTGACTGCCGCCGGCATAACGCATGGAATGGACGAGCTGTATAAAGGCTCCGGTAGTGGTTCCGTCTACGGCAGGAGCATCGCCGTAGGCTTCTGTCTGATGACCGTCGTCCTTCTGCTGGCCGCCGTGATATTCTACTTGAGTTTGGGTGAGTCCTTAGAGTGATGTCCTTTCAAAATTCCATCATTCGCAAACCTAAATAATTTCTGAATCAAGAATGTTCAAAATCTTAGCAATTATTATACGCATAATTTGTGAAACTACTTAAAGTTCTTTTAAAACTTGAGCTGCTGTAAATTTCTATATATACTTTCGTATCCTTAAAGGGTTCCTTCGCTTGAAGCAAAAACCAAAATCAAATTCCAAACTGCAAA-3′5′-CACTTAAAACTCTTCTCCGCGAGCTGTGAACATTAGCCAAATGAAGTGACAAGAAATTAACGCAAAAATAAAACAAGAAGACGGAGCGGTATAAGAAATAATAATATAAAAACTCAATGAGTCAGCACCGCATCAGCTCCTGCTGCTGTTGTTCTTCTTATTGCTGTTGTTTGTGGGGGCGTGGCCGGAGTGGGAATTGAGTGCTCCTAATGATGAACTCGGTCAAGGAGCCAGTGCAGCCATGGTGGCCAAGTAATTAGATAAGCGAGCGTGCAAAACAGGAGCAAACCGATAAATCGCCde novo into a pUC57 plasmid and co-injected with the two gRNAs into the stock y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*], which strongly and ubiquitously expresses a human codon-optimized cas9 . Ten negative hits were also sequenced and two were retained as background controls (ag10 and ag11). The y1M{w[+mC]=Act5C-Cas9.P}ZH-2A w* cassette was removed by selecting against eye colour and stocks were kept as homozygous stocks, except for allele ag8, which was kept balanced over TM6B.These primers were designed to indicate the correct insertion of the sfGFP repair cassette from both sides in the genome. Positive hits were sequenced . The next morning, the flies were transferred to a fresh plate to lay eggs for 3–6 h, depending on the experiment. To control for overcrowding, between 10 and 30 larvae were transferred to vials containing normal Egg collections were performed on normal food plates and larvae were reared at controlled densities without additional yeast . Newly molted third instar larvae were collected every 2 h as described previously48Larvae were collected as described above and transferred 72 h after egg laying to fresh food with 10 mM of EMS (Sigma) or PBS as control. Developmental time was measured as indicated above. EMS food was prepared as follows: food was melted and cooled to 55 °C, an appropriate volume of freshly made EMS stock solution in PBS was added and thoroughly mixed and 3 ml per tube were dispensed. EMS and PBS tubes were kept in the dark as much as possible throughout the experiments.Brains of wandering third instar larvae or first instar larvae were dissected in cold PBS, fixed for 30 min in 4% paraformaldehyde, rinsed with PBS with Triton (0.3%) (PBST), incubated with primary antibody overnight and with fluorescently labelled secondary antibody for 2–24 h in PBST with 1% bovine serum albumin. Nuclei were counterstained with DAPI (Sigma) and tissues were mounted in Fluoromount-G (Southern Biotech). Antibodies used were: mouse anti-GFP 1:200 , mouse anti-nc82 1:250 (DSHB), mouse anti-HA 1:50 , rabbit anti-GFP 1:200 , rabbit anti-Dilp7 1:5000 (gift from I. Miguel-AliagaAleft-Aright/[(Aleft+Aright)/2]. Results were compared statistically using the F-test for the significance of the difference between the FAi of the samples, an appropriate test for dispersion3α/n) for multiple comparisons was applied to α=0.05.Pairs of the left and right wings of male individuals reared at 29 °C and rinsed with ethanol were dissected in a glycerol/ethanol solution and mounted in glycerol. Photos were obtained in a Zeiss Axiovert 40 CFL microscope. The wing areas and wing lengths were calculated as previously described3Lgr3+/+ by a factor of ∼23 .To control for measurement errors, we measured the area of the same wing three times. Values obtained were 18310.2±30.5 a.u., which gives a coefficient of variation (CV) of 0.17%, which is smaller than the CV of wing areas of control −1 (Roche), and incubated the mixture at 37 °C for 1 h, followed by 95 °C for 5 min, to inactivate the protease.gDNA was extracted from a group of flies or single fliesg for 1 min, to lower tissue debris. An extra DNAse treatment  was performed to reduce gDNA contamination. cDNA synthesis was performed using the Maxima First Strand cDNA Synthesis Kit for RT–quantitative PCR (Thermo Scientific), following manufacturer's instructions.RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research), following manufacturer's instructions. The material used for the RT–PCR experiments described in For this study, PCR and RT–PCR primers were designed and their specificity tested using Primer BLAST or Primer3. A T100 Thermal Cycler (Bio-Rad) was used for performing the PCR steps. The following primers were used for PCR analyses described in Lgr3_salto_fw (expected product size 866 bp)Fw: 5′-CCGACGCCTTGCTGCTAACT-3′Rv: 5′-TTTATGGAGCGGGCGTGGTC-3′Lgr3_exonshort Lgr3 (expected product size 331 bp)Fw: 5′-CCGACGCCTTGCTGCTAACT-3′Rv: 5′-GTGCGTTATGAGGTTGTGCTG-3′Lgr3_exon3p Lgr3 (expected product size 240 bp)Fw: 5′-CGCCTTGTCGGTAATCCCAT-3′Rv: 5′-GTGGCTCCATTAAACTGCTGC-3′Lgr3_exons Fw: 5′-CCGACGCCTTGCTGCTAACT-3′Rv: 5′-CAAAGACCACCAACCAGGCGTA-3′rp49 (control)Fw: 5′-TTGAGAACGCAGGCGACCGT-3′Rv: 5′-CGTCTCCTCCAAGAAGCGCAAG-3′qRT–PCR experiments were performed using Lightcycler 96 (Roche) using the FastStart Essential DNA Green Master dye and polymerase (Roche). The final volume for each reaction was 10 μl, consisting of 5 μl of dye and polymerase (master mix), 2 μl of 10 × diluted cDNA sample and 3 μl of the specific primer pairs. The following primers were used:rp49 (control)Fw: 5′-TTGAGAACGCAGGCGACCGT-3′Rv: 5′-CGTCTCCTCCAAGAAGCGCAAG-3′Lgr3Fw: 5′-GCTGGGTGCCCATCATCGTTAT-3′Rv: 5′-CAAAGACCACCAACCAGGCGTA-3′InRFw: 5′-TGTCAGCTGCACAATAATAGGC-3′Rv: 5′-TGCACTTTTCAGGGCATTT-3′DrlFw: 5′-CGGAGTTCCATACCCAGATTAC-3′Rv: 5′-GCCTCTTGTTATATTTACAGGTCTTGG-3′rp49 according to the formula: %rp49=(2̂−(ΔCqLgr3−ΔCqrp49)) × 100. The geometric mean±s.e.m. of three biological repeats was used and data were analysed by one-tailed unpaired Student's t-test using Bonferroni corrections (α/n) for multiple comparisons, using α=0.05. This test assumes the independent samples have equal variances.Primer efficiency for qRT–PCR was tested by serial dilution. Data were expressed as %9Drosophila SL2/DL2 cells (ATCC) cultured in Schneider medium supplemented with 10% foetal bovine serum. The cells were gently oxidized with NaIO4 and TriCEPS–ligand complexes were incubated with the cells for 1.5 h to allow capture of oxidized glycomoieties. Following cell lysis, trypsinization and biotin-mediated affinity purification of ligand–receptor peptides, the glycosylated peptides were selectively released using PNGase F, and samples were analysed by Dualsystems Biotech AG on an LTQ Orbitrap XL (Thermo Scientific) spectrometer fitted with an electrospray ion source. The samples were measured in data-dependent acquisition mode in a 40-min gradient using a 10-cm C18 column. Peptide identifications were filtered to a false-discovery rate (FDR) of ≤1%   and qua014 ref. . For stav2) ref. . The adjIn all experiments reported in this work, no data point was excluded. All data points, including outliers, are represented in the figures and were used in the statistical analyses. No blinding was done and no particular randomization method was used to attribute individuals to experimental groups.Accession codes: DNA sequence data has been deposited in GenBank under accession codes KT321103-KT321113.How to cite this article: Garelli, A. et al. Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing. Nat. Commun. 6:8732 doi: 10.1038/ncomms9732 (2015).Supplementary Figures 1-15 and Supplementary Tables 1-2