A critical step in the removal of polyubiquitinated proteins from macromolecular complexes and membranes for subsequent proteasomal degradation is the unfolding of an ubiquitin moiety by the cofactor Ufd1/Npl4 (UN) and its insertion into the Cdc48 ATPase for mechanical translocation. Here, we present a stepwise protocol for the assembly and purification of Lys48-linked ubiquitin chains that are fluorophore labeled at specific ubiquitin moieties and allow monitoring polyubiquitin engagement by the Cdc48-UN complex in a FRET-based assay.For complete details on the use and execution of this protocol, please refer to Williams et al. (2023).  •Linkage-specific enzymatic synthesis of ubiquitin chains•Ubiquitin labeling with fluorescent dyes•Positional attachment of fluorescent dyes to ubiquitin moieties in polyubiquitin chains•FRET-based assay for ubiquitin unfolding by Ufd1/Npl4 and insertion into Cdc48 Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. A critical step in the removal of polyubiquitinated proteins from macromolecular complexes and membranes for subsequent proteasomal degradation is the unfolding of an ubiquitin moiety by the cofactor Ufd1/Npl4 (UN) and its insertion into the Cdc48 ATPase for mechanical translocation. Here, we present a stepwise protocol for the assembly and purification of Lys48-linked ubiquitin chains that are fluorophore labeled at specific ubiquitin moieties and allow monitoring polyubiquitin engagement by the Cdc48-UN complex in a FRET-based assay. We also describe how chain variants containing a substrate protein or a Met1-linked ubiquitin on the C-terminus of the most proximal ubiquitin are constructed. We used these specifically Cy3-labeled chains together with Cdc48-Ufd1/Npl4 (UN) complexes that had a Cy5 acceptor fluorophore conjugated at a position in the central processing channel of Cdc48 to monitor polyubiquitin-chain engagement in a FRET-based assay. Our studies revealed that Cdc48-UN unfolds and engages ubiquitins in a position- and linkage-specific manner.The protocol below describes the assembly of Lys-48-linked polyubiquitin chains containing a Cy3-donor fluorophore on the N-terminus of the most proximal ubiquitin  competent cells with the expression plasmids for tag-less wild-type S. cerevisiae ubiquitin or ubiquitin with a Met-Cys extension N-terminal to the native Met1 (MCM-Ub) by heat shock at 42°C in a water bath for 45 s. Recover cells for 1 h at 37°C in SOC medium.2.Prepare 50 mL starter cultures in dYT  with the appropriate antibiotic and grow 14 h at 37°C.3.Inoculate 1 L of Terrific Broth containing kanamycin  with 8 mL of starter culture and grow at 37°C while shaking at 180 rpm until OD600 = 1.0.4.Induce protein production for ∼18 h at 18°C with 1 mM IPTG .5.Harvest cells by centrifugation at 3,500 rcf for 30 min at 18°C–20°C and resuspend the resulting pellets in NiA buffer  and clarify the lysate by centrifugation at 27,000 rcf for 30 min at 4°C.7.Add glacial acetic acid to the supernatant until a pH of 4.5 is reached, as monitored by pH paper.8.Stir the solution for 30 min at 18°C–20°C to precipitate proteins other than ubiquitin.9.Clarify the solution by centrifugation at 27,000 rcf for 30 min at 4°C.Note: Soluble ubiquitin will be found in the supernatant.10.Dialyze the supernatant against 4 L of cation buffer A  that has been equilibrated with cation buffer A. Elute ubiquitin from the column with a 0 mM–350 mM NaCl gradient over 20 column volumes using cation buffer A and cation buffer B on a Cytiva ÄKTA Pure FPLC or equivalent  equilibrated in GF buffer  and cysteine-ubiquitin (MCM-Ub-His) were expressed and cells lysed using the same steps 1–6 as above.C-terminally His14.Following lysis, supernatants were incubated with Ni-NTA beads  and batch bound for 30 min.15.Beads were washed with NiA buffer, and proteins were eluted with NiA buffer plus 350 mM imidazole.16.Eluates were concentrated with a 3 kDa Amicon spin filter  and subjected to size-exclusion chromatography using a Superdex 75 16/600 column  equilibrated in GF buffer.CRITICAL: TCEP at 1 mM was included in all purification steps above for MCM-Ub-His to keep the cysteine reduced for subsequent labeling with Cy3-maleimide.Timing: 3 days17.Ube1, the E2-E3 chimera gp78RING-Ube2g2, and PreScission protease were expressed and cells lysed using the same steps 1–6 as above for Ub and MCM-Ub.18.Following lysis, supernatants were incubated with Ni-NTA beads and batch bound for 30 min.19.Beads were washed with NiA buffer, and proteins were eluted with NiA buffer plus 350 mM imidazole.20.6-tag. The high-imidazole buffer was exchanged for NiA buffer by repeated concentration and dilution, and a subtractive step with Ni-NTA beads was used to remove uncleaved gp78RING-Ube2g2 and His-tagged TEV protease.The eluate for gp78RING-Ube2g2 was treated with TEV protease (at a TEV-to-E2-E3 w/w ratio of 1:200) for 14–16 h at 4°C to cleave the N-terminal His21.The flow-through containing gp78RING-Ube2g2 as well as the Ube1 and PreScission elutions from Ni-NTA beads were concentrated with spin filters and subjected to size-exclusion chromatography using a Superdex 75 16/600 (gp78RING-Ube2g2 and PreScission) or a Superdex 200 16/600 (Ube1) column equilibrated in GF buffer.Timing: 2–3 h22.MCM-Ub and MCM-Ub-His at 50 μM in GF buffer were incubated with 1 mM TCEP for 5 min to ensure cysteines are reduced for labeling.23.Sulfo-Cy3-maleimide  was added at a 10-fold molar excess (500 μM) and labeling occurred for 30 min at 18°C–20°C. See 24.A 1 M stock of dithiothreitol (DTT) was added at 5 mM to quench unreacted maleimide dye, and the Cy3-labeled proteins were subjected to size-exclusion chromatography with a Superdex 75 increase 10/300 column equilibrated in GF buffer. 25.Cy3Ub and Cy3Ub-His preparations was calculated by comparing the total protein concentration determined by a Bradford assay and the concentration of Cy3 dye determined by absorbance at the excitation wavelength maximum (555 nm). Labeling efficiency for CRITICAL: Ensure that purified protein stocks are free of thiol-containing contaminants that can react with maleimide. Quenching the labeling reaction with DTT is important, as unreacted Cy3-maleimide can damage chromatography columns.Ub: MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKSTLHLVLRLRGG.MCMUb:MCMQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG.MCMUbHis:MCMQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGLEVLFQGPHHHHHH.D602AzF:Cdc48MGSSHHHHHHSQDPLEVLFQGPMGEEHKPLLDASGVDPREEDKTATAILRRKKKDNMLLVDDAINDDNSVIAINSNTMDKLELFRGDTVLVKGKKRKDTVLIVLIDDELEDGACRINRVVRNNLRIRLGDLVTIHPCPDIKYATRISVLPIADTIEGITGNLFDVFLKPYFVEAYRPVRKGDHFVVRGGMRQVEFKVVDVEPEEYAVVAQDTIIHWEGEPINREDEENNMNEVGYDDIGGCRKQMAQIREMVELPLRHPQLFKAIGIKPPRGVLMYGPPGTGKTLMARAVANETGAFFFLINGPEVMSKMAGESESNLRKAFEEAEKNAPAIIFIDEIDSIAPKRDKTNGEVERRVVSQLLTLMDGMKARSNVVVIAATNRPNSIDPALRRFGRFDREVDIGIPDATGRLEVLRIHTKNMKLADDVDLEALAAETHGYVGADIASLCSEAAMQQIREKMDLIDLDEDEIDAEVLDSLGVTMDNFRFALGNSNPSALRETVVESVNVTWDDVGGLDEIKEELKETVEYPVLHPDQYTKFGLSPSKGVLFYGPPGTGKTLLAKAVATEVSANFISVKGPELLSMWYGESESNIRDIFDKARAAAPTVVFLDELDSIAKARGGSLG∗AGGASDRVVNQLLTEMDGMNAKKNVFVIGATNRPDQIDPAILRPGRLDQLIYVPLPDENARLSILNAQLRKTPLEPGLELTAIAKATQGFSGADLLYIVQRAAKYAIKDSIEAHRQHEAEKEVKVEGEDVEMTDEGAKAEQEPEVDPVPYITKEHFAEAMKTAKRSVSDAELRRYEAYSQQMKASRGQFSNFNFNDAPLGTTATDNANSNNSAPSGAGAAFGSNAEEDDDLYSGSGSGSGSGSGLNDIFEAQKIEWHE.6-TEV-gp78RING-Ube2g2:HisMGSSHHHHHHDYDIPTTENLYFQGHMEARFAVATPEELAVNNDDCAICWDSMQAARKLPCGHLFHNSCLRSWLEQDTSCPTCRMSLNIADNNRVREEGTGSHMAGTALKRLMAEYKQLTLNPPEGIVAGPMNEENFFEWEALIMGPEDTCFEFGVFPAILSFPLDYPLSPPKMRFTCEMFHPNIYPDGRVCISILHAPGDDPMGYESSAERWSPVQSVEKILLSVVSMLAEPNDESGANVDASKMWRDDREQFYKIAKQIVQKSLGL.Timing: 2–3 hCy3Ub-Ubn). The proximal ubiquitin is the ubiquitin closest to the substrate and in case of unanchored chains has a free C-terminal glycine, while the distal ubiquitin extends away from the substrate and represents the last moiety added to a ubiquitin chain . For studies with Cdc48-Ufd1/Npl4, we aimed to generate heterogenous chains centered around Ub5 in length. The C-terminal His-tag on unlabeled MCM-Ub-His serves the same purpose as in the major step above. Only unlabeled Ub will be present at the most proximal position in a chain. Formation of Ub-Cy3Ub-Ubn, however, requires two steps. In this first step, limiting Cy3Ub is added to MCM-Ub-His to form Ub-Cy3Ub and small amounts of Ub-Cy3Ub-Cy3Ub. After a Ni-NTA purification step, Ub-Cy3Ub is mixed with unlabeled Ub to form Ub-Cy3Ub-Ubn.8.Cy3Ub, 1 μM E1 and 20 μM of the E2-E3 chimera, gp78RING-Ube2g2, in ubiquitination buffer.Create a mixture of 50 μM Ub-His, 5 μM 9.Cy3Ub and unreacted Ub-His. Initiate elongation by adding ATP to 10 mM. Ideally, the major products will be Ub-CRITICAL: The goal of this part of the protocol is to add limiting amounts of Cy3Ub to the ubiquitination reaction such that the major product is elongation of Ub-His by a single addition of Cy3-labeled ubiquitin (to form Ub-Cy3Ub). Conditions that lead to elongation of Ub-His by multiple additions of Cy3-labeled ubiquitin Cy3Ub (forming Ub-Cy3Ubn) should be avoided. Small-scale test reactions can be performed using different molar ratios of Ub-His to Cy3Ub to determine optimal ubiquitination reaction conditions  in assay buffer  with and without 4 μM UN in assay buffer supplemented with 1 mg/mL BSA and 1 mM ATPγS. These are the 2X Cy5Cdc48 and Cy5Cdc48-UN samples.Create samples of 4 μM 17.Cy5Cdc48 or Cy5Cdc48-UN sample. Incubate at 30°C in a thermocycler. The final concentrations are 0.2 μM Cy3Ub-Ubn or Ub-Cy3Ub-Ubn, 2 μM Cy5Cdc48, 0 μM or 2 μM UN, and 0.5 mg/mL BSA.Create mixtures consisting of 6 μL of the 2X polyubiquitin sample and 6 μL of the 2X Note: Pore-insertion kinetics are rapid (∼3 s)ref.18.Transfer 10 μL of each mixture into wells of a 384-well black plate  pre-heated at 30°C and load into a microplate reader (BMG Labtech CLARIOstar Plus) equipped to measure fluorescence emission spectra of Cy3 and Cy5 following excitation at the absorbance maximum for Cy3.19.Incubate at 30°C and scan wells for fluorescence emission between 525 nm and 725 nm using an excitation wavelength of 480 nm CRITICAL: Since both Cy3Ub-Ubn and Ub-Cy3Ub-Ubn contain contaminating unlabeled polyubiquitin chains, pore-insertion experiments are performed with a 10-fold excess of Cy5Cdc48-UN to avoid any competition between labeled and unlabeled chains. However, these conditions lead to technical challenges due to some extent of direct Cy5-acceptor excitation when exciting the Cy3 donor, which is further exacerbated by the fact that Cdc48 homohexamers can be modified with multiple Cy5 fluorophores depending on the labeling conditions and efficiency. If there is excessive direct excitation of Cy5, the Cy5 emission peak will dominate the spectrum and, due to spectral overlap, introduce some background fluorescence in the region of the Cy3 emission peak as well. To minimize Cy5 fluorescence emission resulting from direct excitation, experiments should be performed with an Cy3-donor excitation wavelength of 480 nm or lower.20.Cy5Cdc48 and UN  are synthesized in a single step in which unlabeled ubiquitin is added progressively to Cy3-labeled ubiquitin with a C-terminal His-tag in the presence of ubiquitination machinery. The C-terminal His-tag on the fluorophore-labeled species is critical, because it restricts the labeled ubiquitin to the first position in the chain by preventing conjugation of this moiety to itself or to other ubiquitins during the course of the reaction. A tight distribution of polyubiquitin chain lengths is expected  are synthesized in two steps. In the first step, a limiting amount of Cy3-labeled ubiquitin is added to unlabeled ubiquitin whose C-terminus is blocked with a His6-tag to yield a ubiquitin dimer with the labeled species in the second position (Ub-Cy3Ub). The molar ratio of these species should be optimized such that the single addition of Cy3-labeled ubiquitin to unlabeled ubiquitin is the major product  that can react with maleimide conjugated fluorophores. See Perform a dialysis or desalting step with a 7K MWCO spin desalting column  to exchange for buffer compatible with maleimide labeling.Following conjugation, my cysteine-ubiquitin sample did not elute from the size-exclusion chromatography column or eluted in the void peak. See Attachment of hydrophobic fluorophores to ubiquitin can greatly alter its properties and lead to aggregation or adsorption to filter membranes. We have achieved the best results with sulfonated fluorophores that are more hydrophilic and water soluble. The stability of the fluorophore-conjugated ubiquitin can also depend on the labeling position. Ubiquitin labeled at an N-terminal cysteine may be more well-behaved than ubiquitin labeled at an engineered internal cysteine.My fluorophore labeling efficiency is low. See Ensure that the cysteine-containing ubiquitin sample is in a buffer free of thiol-containing reagents. Oxidation of cysteine during the purification steps prior to conjugation will decrease the labeling efficiency. Include a reducing agent  throughout the purification to keep cysteines reduced.Cy3Ub-His, a diffuse distribution of chains was formed. See During the first round of chain extension on Decrease the amount of unlabeled ubiquitin in the reaction. If the concentration of unlabeled ubiquitin is too high, the processive E2-E3 ligase gp78RING-Ube2g2 will form a broad length distribution and distinct bands will not be visible on the gel.Cy3Ub is a ubiquitin trimer (Ub-Cy3Ub-Cy3Ub) or longer chain (Ub-Cy3Ub-Cy3Ubn). See The major product of the elongation of Ub-His with Cy3Ub to Ub-His.As shown in the example gel A, the maThe Cy3 fluorescence emission peak is masked by the Cy5 fluorescence emission peak originating from direct excitation of excess Cy5 fluorophores. See Cy5Cdc48 could be used in the assay to decrease the size of the Cy5 emission peak, but doing so may compromise the extent of donor quenching if the motor is no longer at concentrations that are saturating for polyubiquitin-chain binding. Another potential solution is to decrease the number of Cy5 fluorophores conjugated to Cdc48 hexamers by performing labeling reactions with lower concentrations of Cy5-maleimide.A lower concentration of a.martin@berkeley.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Andreas Martin (This study did not generate new unique reagents.


AbstractPageBreakCatharylla Zeller, 1863  is redescribed. Catharylla contiguella Zeller, 1872, C. interrupta Zeller, 1866 and Myelois sericina Zeller, 1881, included by Munroe (1995) in Catharylla, are moved to Argyria Hübner. Catharylla paulella Schaus, 1922 and C. tenellus  are redescribed. Six new species are described by Léger and Landry: C. bijuga, C. chelicerata, C. coronata, C. gigantea, C. mayrabonillae and C. serrabonita. The phylogenetic relationships were investigated using morphological as well as molecular data . The median and subterminal transverse lines of the forewing as well as the short anterior and posterior apophyses of the female genitalia are characteristic of the genus. The monophyly of Catharylla was recovered in all phylogenetic analyses of the molecular and the combined datasets, with three morphological apomorphies highlighted. Phylogenetic analyses of the morphology of the two sexes recovered three separate species groups within Catharylla: the chelicerata, the mayrabonillae, and the tenellus species groups. The possible position of Micrelephas Schaus, 1922 as sister to Catharylla, based on both morphological and molecular data, and the status of tribe Argyriini are discussed. The biogeographical data indicate that the chelicerata species group is restricted to the Guyanas and the Amazonian regions whereas the tenellus group is restricted to the Atlantic Forest in the South-Eastern part of Brazil. The mayrabonillae group is widespread from Costa Rica to South Bolivia with an allopatric distribution of the two species. COI barcode sequences indicate relatively strong divergence within C. bijuga, C. mayrabonillae, C. serrabonita and C. tenellus.The Neotropical genus  Pyraloidea is one of the largest superfamilies of the order Lepidoptera. The monophyly of the group and that of its two main lineages, the Pyraliformes and Crambiformes , are well supported by morphological characters , but these share no evident common characteristics other than the snow white color of the wings.Catharylla is revised using both morphological and molecular data. Phylogenetic relationships within Catharylla and with putatively related taxa as well as the distribution of each species along with biogeographical hypotheses are analysed.In this work, PageBreakCatharylla and outgroup taxa investigated were borrowed from museums and private collectors as listed in Several of the MHNG specimens were kindly given to this institution by collaborators mentioned in the acknowledgments section. The specimens were usually well preserved, the color being sometimes faded. Many specimens from the BMNH were dissected by S. Bleszynski. Unfortunately, his preparations were usually poorly made and badly mounted, sometimes hampering the investigation of genitalia characters.The dissection and slide mounting methods follow Catharylla species, four additional crambine species were included in the dataset for phylogenetic analyses. The material investigated to build the morphological matrix is reported below in Apart from the eight PageBreakPageBreakCatharylla contiguella Zeller, 1872, and Catharylla interrupta Zeller, 1866 could not be found. With the help of the descriptions and illustrations, they were excluded from Catharylla because of the forewing pattern, which is like that of Argyria Hübner, 1818, with only one median transverse line. For Catharylla sericina Zeller, 1881, based on the description and a photograph of the type in the BMNH, the species was rejected from Catharylla based on the elongated forewing shape and the silvery white pattern without transverse lines. For Catharylla paulella Schaus, 1922, a photograph of the habitus and the genitalia of the female type from the USNM allowed to find other specimens of the same species; the male and female were then associated based on wing pattern. For Catharylla tenellus Zeller, 1839, a photograph of the habitus and the genitalia of the female type were available and the male of the species was associated based on wing pattern. For the descriptions, we followed the nomenclature and terminology used by The types of the two species The following measurements were made with the use of an ocular micrometer: length of labial palpus (base of segment I to apex of segment III), diameter of eye , length of forewing (from base to apex), length of uncus (from tegumen-uncus junction to apex of uncus), length of tegumen connection , length of papillae anales , lengths of anterior and posterior apophyses (from base to apex).Regarding the holotype data, the information was copied exactly as found on the labels with vertical slashes to express changes of lines. Abbreviations are spelled out in square brackets. We assume that the labels are rectangular and white, and that the text is in black ink, otherwise differences are indicated in brackets. Paratype data are reported by country in alphabetical order and the information is recorded without indication of line change. Collecting localities are reported as written on labels, with a question-mark when the locality could not be recovered. Dates and collectors’ information were standardized and the latter placed in parentheses. The specimen depositories are reported with the use of the corresponding acronyms.PageBreakhttp://earth-info.nga.mil/gns/html/). The localities were reported on a text file (*.txt) and loaded on a map using DIVA-GIS 7.4.0.1 . The localities that were not registered in Google Earth were localized more or less precisely with the help of internet search engines or with gazeeters from the GEOnet Names Server (GNS) of the National Geospatial Intelligence Agency  and the two nuclear genes wingless (353 bp) and EF-1α (679 bp). These genes show different rates of substitution through time, with COI >> wingless > EF-1α . COI peridoptera .Specimens used for molecular investigations are listed in Catharylla specimens collected no more than twenty years ago, a leg was sent to the Canadian Centre for DNA Barcoding (CCDB) at the Biodiversity Institute of Ontario (BIO) in Guelph. The barcode sequences of Catharylla are reported in each species description. The protocol for DNA extraction is found in the supplementary material of http://dev.ccdb.ca/docs/CCDB_Amplification.pdfFor DNA extractions were performed following the method of The primers used are recorded in PageBreakPageBreakPageBreakPCRs were performed using peqGOLD Taq DNA polymerase (PeqLab). In cases of weak or absent PCR result, a re-examination PCR was done using BIO-X-ACT Short Taq polymerase (Bioline). PCR protocols are given in Success of gene amplification was evaluated by an electrophoresis with 1% agarose gel, subsequent gel dying with GelRed and analysis under ultraviolet light. PCR products were purified using ExoSAP-IT (USB Corporation).Sequence PCR was done with the BigDye Terminator-Kit of Applied Biosystems. The amount of each product is reported in Sequence alignment was done with BIOEDIT 7.1.3  and PhyDhttp://www.ebi.ac.uk/ena/data/view/HG793012-HG793020; http://www.ebi.ac.uk/ena/data/view/HG793012-HG793020PageBreakGenetic distances between barcoding sequences of COI are given in Crambus pascuella and Crambus uliginosellus set as outgroups. Maximum parsimony analyses were performed using the Branch-and-bound method as searching algorithm with parameters left unchanged. The bootstrap resampling method with 1000 replicates was used. The resulting tree was a 50% majority-rule consensus tree with bootstrap (BS) values assigned to each node. BS supports are reported on the tree of The 21 characters are listed in PageBreakCatharylla bijuga and Catharylla coronata, and the whole COI gene for Catharylla paulella. For Catharylla bijuga, we used the sequence BC MTD 1840 to build the datasets as this sequence performed better than BC MTD 1839 in phylogenetic analyses. The COI sequence of LEP 972 was combined with that of wingless for LEP 973 given that the two samples come from the same population and are genetically very similar as attest the barcode sequences. We generated four different datasets from the sequence data: a complete dataset with all three genes sequences available for the 12 taxa (mol_1), a 12-taxa dataset with the 3rd codon position of COI deleted (mol_2), a dataset restricted to the 7 taxa for which the sequence of the COI gene and at least of one nuclear gene were available (nucl_1), and the same data as nucl_1 but with the 3rd codon position of the COI deleted (nucl_2). We used the programme RAxML  over 0.95 are considered well supported. PP values are reported on the tree  the morpho-matrix , (2) COIthe tree .Zeller, 1863http://species-id.net/wiki/CatharyllaCatharyllaCrambus tenellus Zeller, 1839, by subsequent designation by PageBreakCatharylla species have snow white to creamy white wings and short labial palpi. They can be separated from other Argyriini by the presence on the forewing of median and subterminal thin transverse lines, slightly curved, convex on costal 1/3. The labial palpi are also shorter in comparison to those of Vaxi. The highly variable male genitalia do not show any synapomorphy or generic diagnostic character. In females, a possible synapomorphy is the strongly reduced anterior and posterior apophyses of abdominal segments VIII and IX, but this is shared with some Crambini and a few other Crambinae (see inae see .Catharylla chelicerata)  : R1 presicerata)  backgrouTympanal organs : TransveCatharylla tenellus group, strongly developed. Juxta medially curved downward, narrowing toward rounded apex, slightly directed downward apically, basally triangular, sometimes with additional lobes at base or ventro-lateral projections. Vinculum of medium width; saccus short, rounded, directed anterad and slightly upward. Phallus straight or curved, usually more strongly sclerotized at apex; vesica without cornuti, with one cornutus, or with crest of cornuti.Male genitalia –33: UncuPageBreakrior apophyses 0.25–0.45 × length of papillae anales, straight, regularly thin. Tergite VIII narrow, with postero-dorsal line of setae. Anterior apophyses reduced, 0.01–0.1 × length of papillae anales. Sternite VIII about twice length of tergite, not connecting ventrally in tenellus species group. Sterigma present, strongly sclerotized except in tenellus species group, usually forming pockets of variable shape; reduced to sclerotized lamella antevaginalis in Catharylla bijuga. Ductus seminalis connecting posteriorly at base of ductus bursae. Ductus bursae long, at least 2 × length of corpus, wide, basally curved. Corpus bursae usually rounded, egg-shaped, often enlarging progressively from ductus bursae, usually with one signum, sometimes without; corpus and ductus bursae covered with minute spicules.Female genitalia : PapillaThe genus is restricted to the Neotropical Region, from Costa Rica to Santa Catarina, Brazil, from sea level to 1300 m –46.Catharylla serrabonita in its environment, i.e. forested hills up to about 950 m in elevation, surrounded by cacao or coffee plantations in the lowlands. The moths were coming to light, usually very late (after 23:00).The biology of the species remains unknown. In the Serra Bonita Reserve in march 2011, we observed Catharylla has been placed in the Argyriini , Amazonas, P[ar]q.[ue] Nac.[ional] do Jaú, Rio Jaú, bg. Miratucú, 1°57'S, / 61°49'W, 26–27.vii.1995, U[ltra] V[iolet] light sheet (R. W. Hutchings) (USNM). FRENCH GUIANA: 2 ♂  with same data as holotype (BMNH); 5 ♂, 1 ♀  with same locality as holotype except i.1918 (1 ♂), ii.1918  (S. M. Klages) ; 1 ♂, 2 ♀, Parcelles CIRAD de Combi, plantations expérimentales pk 1.5, 5°18'N, 52°55'30 W, 4.iii.2011, piège lumineux [light trap] (B. Hermier)  (MHNG); 3 ♂, 2 ♀ , Saint-Jean-du-Maroni (Le Moult) (BMNH); PageBreak1 ♂, Saint-Laurent-du-Maroni (USNM); 1 ♂  Roura, 3.6km E[ast] Roura at r[oa]d to Crique Gabrielle, 50m, 20.iv.1994, at light (J. S. Miller & C. Snyder) (AMNH); 1 ♀ , Cayenne, iii.1917 (CMNH). GUYANA: 1 ♂, New River 1938 (C.A. Hudson) (BMNH); 1 ♂, Mallali [sic] (USNM). SURINAME: 1 ♀ , Geldersland, Surinam River (USNM); 1 ♀ , Sipaliwini Distr[ict], Thibiti area, Kabo Creek, partly swampy, primary forest on hilly slopes, ca. 2km from river, 29.v.1989 (J. Beerlink) (Schouten Coll.).COI barcode sequence of paratype BC MTD 01839 (654 bp): ACATTATATTTTATCTTCGGAATTTGAGCAGGAATAGTTGGAACATCCCTAAGACTTTTAATTCGAGCAGAATTAGGTAATCCAGGTTCTCTTATTGGTGACGACCAAATTTATAATACTATTGTTACTGCTCATGCATTTATTATAATTTTTTTTATAGTTATGCCAATTATAATTGGAGGATTCGGTAATTGATTAGTTCCATTAATATTGGGAGCACCAGATATAGCATTCCCACGAATAAATAATATAAGATTTTGATTACTCCCCCCCTCTTTAATCCTATTAATTTCTAGAAGAGTTGTAGAAAATGGAGCTGGAACAGGATGAACAGTTTACCCCCCACTTTCATCAAATATTGCTCATAGTGGTAGATCTGTAGATTTAGCAATTTTTTCTCTACACTTAGCAGGAATTTCATCAATCTTAGGAGCTATTAATTTTATTACAACAATTCTTAATATACGAATTAATGGTTTATCTTTCGATCAAATACCTTTATTTGTTTGATCTGTAGGAATTACAGCTTTACTTCTTCTCTTATCCTTACCCGTATTAGCTGGTGCTATTACTATACTTTTAACTGATCGAAATTTAAATACATCTTTTTTTGATCCTGCTGGAGGAGGAGATCCTATCCTTTACCAACACTTAOn the forewing , the sevPageBreakunderside dull white to light ochreous along costal margin, with marginal dashes pronounced. Hindwing snow white, veins slightly ochreous, with shiny aspect; marginal PageBreakline thin, brown, pronounced up to CuA1, then shiny white; fringes white; underside white, with same margin as on recto.Male (n = 17) : AntennaTympanal organs (n = 8): Tympanic pockets extending slightly beyond transverse ridge, rounded. Tympanic drum elongate, more or less oval, postero-laterally extended beyond transverse ridge.Male genitalia (n = 8) , 12: UncFemale (n = 10): Labial palpi: 1.1–1.4 mm long. Forewing length: 11–15 mm; frenulum triple.Female genitalia (n = 6) : PapillaThe species occurs in lowlands in the three Guianas and Brazil .bijugus, a, um which means “yoked together, double”, in reference to the bifid costal arm of the male genitalia.Bijuga comes from the Latin PageBreakCatharylla, he gave the manuscript name Catharylla ramona to this species, but never published it. The comparison of the tip of the tubular costal arm of the male genitalia and the female lateral projections of sternite VIII shows rather nicely that the male hooks the female genitalia during the mating process. The specimen collected in Parque Nacional do Jaú, Brazil, shows a divergence in COI barcode sequence of 5.05% with that of Roura, French Guiana. In morphology we find no significant difference corroborating this divergence. The relationships of this species to the others remain uncertain in our phylogenetic analyses.In some paratypes from French Guiana the collecting data mention a “pk” (=”point kilométrique”). This kilometric marker refers to the distance of the collecting spot on the forest road to the nearest main road. CIRAD  refers to the name of the research institution leading agronomical research on the Combi site. the Combi site. When S. Bleszynski looked into Diagnosis. The synapomorphies of the group are the quadrangular valva with a truncated apex and the hook-shaped gnathos in the male genitalia. The chelicerata species group can be separated from the other Catharylla species based on additional diagnostic characters. Externally, the forewing has a clear, dark brown costal band, and its length is usually over 14 mm. In male genitalia, the apex of the uncus is regularly rounded with a short, narrow projection medially and the vesica shows one large, curved, pointed cornutus, preceded by a string of 13–14 smaller cornuti increasing in size toward apex. In female genitalia, the ductus bursae shows a pronounced, tongue-shaped projection postero-ventrally.Notes. This group includes two species. The phylogenetic analyses restricted to the nuclear genes and the combined Bayesian analysis place the group as sister-group of the mayrabonillae species group , Amazonas, Rio Negro, Mirapinima, 8.iv.1972  (CNC); 9 ♂, 1 ♀, Reserva Ducke, km 26 Manaus–Itacoatiara Highway, 15.iv.1972 (1 ♂), 18.iv.1972 , 21.iv.1972 , 16.v.1972 (2 ♂), 17.v.1972 , 18.v.1972   (CNC). FRENCH GUIANA: 5 ♂, 1 ♀, 36km SE, Roura (Camp Patawa), 21.xi.2007 , 29–30.xi.2007 , 30.xi.2007 (1 ♂) (MHNG); 1 ♀  with same data as holotype; 1 ♂, same data as holotype except 2.ix.2011 (Hermier n° 24755); 1 ♀, same data as holotype except /604/ and 4.iii.2011 (Hermier n° 24344); 2 ♂, Beauséjour, N[ationale] 1 pk 28.5, 4°42'30"N, 52°23'30"W, 3.vi.2011, piège lumineux (Hermier n° 24545 & 24546) (B. Hermier) (MHNG); 1 ♂, Route d’Apatou pk 25.5 spk 2+4.4, 1.x.2011, piège lumineux (B. Hermier) (Hermier n° 24956) (MHNG); 1 ♂ , R[ou]te forestière de Saut Léodate pk 4.5, 4°55'N, 52°33'W, 31.x.1995 piège lumineux (B. Hermier) (Hermier n° 8457) (MHNG).PageBreakOther specimens. 1 ♀ , Nova Olinda, Rio Purus, v.1922 (S. M. Klages) (CMNH); 1 ♀ , Teffé [sic], vi.1906 (W. Hoffmanns) (BMNH).COI barcode sequence of paratype BC MTD 01703 (654 bp): ACTTTATATTTTATCTTTGGAATTTGAGCAGGAATAATTGGAACATCCTTAAGACTACTAATTCGAGCAGAATTAGGTAATCCTGGATCTCTTATCGGGGATGACCAAATTTATAACACTATTGTTACTGCTCATGCATTTGTAATAATCTTTTTTATAGTTATACCAATTATAATTGGTGGATTTGGAAACTGATTAGTACCTTTAATGCTAGGGGCACCAGATATAGCATTCCCTCGTATAAATAATATAAGATTTTGACTTCTTCCCCCCTCTTTAACCCTATTAATTTCAAGTAGAATTGTAGAAAATGGGGCAGGAACAGGATGAACCGTTTATCCACCTTTATCATCTAATATTGCCCATGGAGGCAGATCAGTAGATCTGGCAATTTTTTCACTACATTTAGCTGGAATTTCATCAATTTTAGGGGCAATTAATTTTATTACAACAATTATTAATATACGAATTAATAATCTTTCATTTGATCAAATACCCCTATTTGTTTGATCAGTAGGTATTACAGCATTACTATTACTTCTATCTTTACCAGTATTGGCGGGAGCTATTACCATACTTCTAACTGACCGAAATCTCAATACTTCCTTTTTTGATCCAGCAGGGGGGGGAGACCCTATTTTATATCAACACCTACatharylla gigantea, Catharylla chelicerata differs in having the male costal arm hook shaped, longer, and thinner than in Catharylla gigantea, and the juxta is strongly downcurved, apically conical whereas it is long, almost straight, without apical conical projection downward in Catharylla gigantea. In female genitalia the sterigma forms a strongly sclerotized symmetrical structure made of two asymmetrical bell-shaped cavities, opened anterad in Catharylla chelicerata whereas it forms a pair of shallow pockets opened posterad in Catharylla gigantea.From Male (n = 21) , 9: HeadPageBreaksclerotized, dorsal base of praecinctorium sclerotized. Tympanic drums elongate, bean shaped, posteriorly reaching transverse ridge or slightly beyond.Tympanal organs (n = 9): Transverse ridge almost straight medially. Tympanic pockets conical, extending slightly beyond transverse ridge. Tympanic bridge lightly PageBreakshaped, forming angle of about 100° with axis of basal arms, about 1/4 longer than uncus. Tegumen arms narrow at base, enlarging progressively toward dorsum to 2 × basal width, projected dorsally with bump at connection, connecting at distal 1/6. Cucculus densely setose, slightly directed upward on distal 1/3, apically truncated; basal 2/3 of costa of valva dorso-ventrally and laterally widened; costal arm hook-shaped, PageBreakPageBreakstrongly sclerotized, directed upward at about 45° from costal arm base. Juxta triangular, curved downward, tip rounded and sac-like, basal lateral lobes curved ventrally. Saccus short, curved upward medially. Phallus narrow, S-shaped; vesica covered with tiny spicules, with one large, curved, pointed cornutus apically, preceded by string of 13–14 smaller cornuti increasing in size toward apex.Male genitalia (n = 9) , 14: UncFemale (n = 4): Labial palpi: 1.8–2.2 mm long. Forewing: 15–19.5 mm. Frenulum quadruple.PageBreakPageBreakanterior margin; covered with minute punctuation. Ventro-basal section of ductus bursae tongue shaped, strongly sclerotized; ductus bursae long, ventrally sclerotized, widened and looped in basal half; enlarging progressively into corpus bursae. Corpus bursae egg-shaped with one signum.Female genitalia (n = 3) : PapillaThe species was found in French Guiana and Brazil (Amazonas) .Chelicerata” refers to the shape of the costal arms of the male valva, which look like mygalomorph chelicerae.“Catharylla robustella  and Catharylla tenellina  by S. Bleszynski, as indicated on labels, but these names were never published. These two specimens are probably Catharylla chelicerata, but the bad genitalia preparations do not allow to see details, and therefore they are not included as paratypes.Two females included here have been named T. Léger & B. Landrysp. n.http://zoobank.org/06B7837B-A5DE-4347-824E-B74127F028E2http://species-id.net/wiki/Catharylla_giganteaHolotype. ♂, with labels as follows: “Brazil: Amazonas, Manaus, | Reserva Ducke, AM-010, k[ilo]m[eter]. 26 | 2°55'S, 59°59'W, Dec[ember].13, 1993 | J. Bolling Sullivan & | Roger W. Hutchings | U[ltra]V[iolet] Light (Plateau Hut)”; “HOLOTYPE | Catharylla gigantea | T. Léger & B. Landry” [red label]; “BL 1747 ♂” [light green label]. Deposited in USNM.Paratypes. 5♂, 2 ♀. BRAZIL: 1 ♂, Amazonas, Reserva Ducke, km. 26, Manaus–Itacoatiara Highway, 15.v.1972  (CNC). FRENCH GUIANA: 1 ♂, 1 ♀ , Saint-Jean-du-Maroni (E. Le Moult) (BMNH); 1 ♂ , Oyapok [sic] River, Pied Saut, iii.1918 (S. M. Klages) (CMNH). GUYANA: 2 ♂, 1 ♀ , Potaro, ii.1908 (2 ♂), v.1908 (1 ♀) (S. M. Klages) (BMNH).Catharylla chelicerata, Catharylla gigantea differs in having the male costal arm shorter, basally wide and tooth shaped while it is long, narrow throughout and hook shaped in Catharylla chelicerata. The juxta is long, tongue shaped, almost straight, and apically rounded, whereas it is downcurved and apically conical in Catharylla chelicerata. In female genitalia, the sterigma forms a pair of shallow pockets opened posterad whereas in Catharylla chelicerata the sterigma forms a strongly sclerotized symmetrical structure made of two asymmetrical bell-shaped cavities opened anterad.From PageBreakbrown scales at half of length, white tipped. Thorax with some brown at collar. Foreleg coxa white, femur white, ashen brown dorsally; tibia and tarsomeres brown-ochreous, distally ringed with dark brown. Midleg white with tibia-femur joint and base of tibia ashen; tarsomeres ochreous to brown ochreous with upperside brown to dark brown, white tipped. Hindleg white with tarsomeres II–V ochreous to brown ochreous, upperside brown, with white tips. Forewing length: 13.5–14.5 mm; snow white with wide brown to dark brown costal line from base to apex; median and subterminal transverse lines faded brown; dark brown spots on termen forming more or less continuous line; fringes brass colored; underside white, with costal margin brown ochreous, outer margin with subtriangular spots. Hindwing snow white; marginal spots dark brown between R5, M1, M2, M3, and CuA1; fringe white; underside snow white, with same spots as on upperside.Male (n = 6) : Head wiTympanal organs (n = 5): Transverse ridge medially convex. Tympanic pockets extending slightly beyond transverse ridge, rounded. Tympanic bridge lightly sclerotized, dorsal base of praecinctorium sclerotized. Tympanic drums elongate, bean shaped.Male genitalia (n = 5) , 16: UncFemale (n = 2): Labial palpi: 2.5–3.1 mm long. Forewing length: 17.5–22 mm; frenulum triple.Female genitalia (n = 2) : PapillaCatharylla gigantea has been found in French Guiana, Guyana, and Brazil (Amazonas) (mazonas) .giganteus, a, um meaning very large.The name comes from the Latin The name was given to the species on manuscript labels by S. Blezynski, probably in reference to the large size of the female.PageBreakDiagnosis. The synapomorphies of the group are the dorsal furrow on the uncus, the uncus apex slightly bifid, the presence of a transtilla in male genitalia, and the absence of a ventral connection of sternite VIII in female genitalia. The tenellus species group can also be separated from the other Catharylla species based on the following additional diagnostic characters: the hindwings are creamy-white, and in female genitalia, the papillae anales are not produced.Notes. This group includes three species, including two new ones. Catharylla serrabonita and Catharylla tenellus form a monophyletic group within the tenellus species group http://species-id.net/wiki/Catharylla_tenellusCrambus tenellus Zeller, 1839: 174–175Catharylla tenella : Argyria tenella : Platytes tenella : Holotype. ♀, “Type” [red ringed]; “Catharylla | tenella Z[eller]. | Mon[ograph]. p[age]. 50 Am. anftr.” [not clearly readable]; “Zell[er]. Coll[ection]. | 1884”; “♀ | Pyralidae | Brit[ish]. Mus[eum]. | Slide N° | 1094”. Deposited in BMNH.Other specimens examined. 20 ♂, 7 ♀. BRAZIL: 3 ♂ , São Paulo, Ubatuba, Picinguaba, 23°22'S, 44°50'W, 2–20m, 22–24.ix.2001 (V. O. Becker n°132820) (Becker Coll.); 2 ♂ with same data except 10–12.xi.2001 (V. O. Becker n°133712) (Becker Coll.); 2 ♂, 1 ♀ , São Paulo, Bertioga, 5 m, 5.xi.1995 (V. O. Becker n°99090) (USNM); 1 ♂  with same data (Becker Coll.); 1 ♂  with same data except 15–17.v.1996 (V. O. Becker n° 99386) (Becker Coll.); 1 ♂ with same data except 7–9.x.1996 (V. O. Becker Coll. n°99757) (Becker Coll.); 1 ♂ , São Paulo, São Luiz do Paraitinga, 23°20'S, 45°06'W, 900 m, 13–20.iii.2001 (V. O. Becker n°132356) (Becker Coll.); 1 ♂ , São Paulo, 700 m (E. D. Jones) (BMNH); 1 ♂ , Minas Gerais, Caraça, 1300 m, 1–2.iv.1992 (V. O. Becker n°85081) (USNM); 1 ♀  with same data (Becker Coll.); 1 ♂  with same data except 25.x.1994  (Becker Coll.); 1 ♂, 1 ♀ , Bahia, Porto Seguro, A. d’Ajuda, 16°27'S, 39°03'W, 20 m, 12.vii.2009 (V. O. Becker n°144140) (Becker Coll.); 2 ♂  with same data except 15.viii.2008 (V. O. Becker n°140808) (Becker Coll.); 1 ♂  with same data except 1–3.v.2009 (V. O. Becker n°142784) (Becker Coll.); 1 ♂, Paranà, Castro (USNM); 1 ♀ , Rio de Janeiro, xi[day and year data missing] (H. H. Smith) (CMNH); 1 ♀ , Rio de Janeiro, Corcovado, 457 m, 26.xii.1958 (E. P. Wiltshire) (BMNH). No locality data: 1 ♂, 1 ♀ ; 1 ♀ , 1869 (NMW).COI barcode sequence of specimen BC MTD 1710 (654 bp): ACTCTATATTTTATCTTTGGAATTTGATCAGGAATAATTGGAACATCTTTAAGATTATTAATTCGAGCAGAATTAGGGAATCCTGGATCTCTAATTGGAGATGATCAAATTTATAACACTATTGTAACAGCCCATGCATTTATTATAATTTTTTTTATGGTTATACCAATTATAATTGGTGGATTTGGAAATTGATTGGTTCCATTAATATTAGGAGCCCCAGATATAGCTTTCCCCCGAATAAATAACATAAGATTTTGGTTATTACCCCCTTCCTTAACTCTTTTAATTTCTAGAAGAATTGTAGAAAATGGAGCTGGAACAGGATGAACGGTCTACCCCCCCCTTTCATCTAATATTGCCCATAGTGGAAGATCTGTAGATTTAGCAATCTTTTCTCTTCATTTAGCTGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAATATACGAATTAGTAATTTATCTTTTGATCAAATACCTTTATTTGTTTGATCAGTCGGTATTACAGCTTTACTTCTTCTTCTATCTTTACCTGTATTAGCAGGAGCTATTACTATACTTTTAACTGATCGAAATTTAAATACATCTTTTTTTGATCCTGCAGGAGGAGGAGATCCTATCTTATATCAACATTTACatharylla serrabonita and Catharylla coronata, Catharylla tenellus can be separated by the median transverse line, which is faintly convex towards costa, whereas it is more strongly convex in Catharylla coronata and Catharylla serrabonita. The male genitalia provide the best diagnostic characters. The most obvious refers to the transtilla, which forms a pair of short, narrow sclerotized arms with pointed tips, projecting posterad, with, in between, a pair of brushes directed medio-ventrally, whereas it forms a pair of arms pointing posterad with a string of spines ventrally in Catharylla serrabonita and Catharylla coronata. In female genitalia, the anterior angle of sternite VIII is directed downward into a more or less rounded projection covered with short spinules of same length, whereas it is projected anterad in Catharylla serrabonita, and it is not projected in Catharylla coronata.From PageBreaktips. Thorax slightly ochreous at collar. Foreleg coxa white; femur ochreous, dorsally dark brown; tibia and tarsomeres ochreous, distally ringed with dark brown. Midleg light ochreous with tibia-femur joint brown; tarsomeres II–V dark brown on upperside, with white ringed tips. Hindleg white; tarsomeres as midleg. Forewing length: 10.5–12 mm; snow white; costal line ochreous, lightly pronounced from base to apex; median and subterminal transverse lines ochreous, median transverse line faintly convex towards costa; outer margin ochreous with 7 dark brown spots often triangular, strongly pronounced; fringes brass colored; underside ochreous with costal margin pronounced in basal half and marginal spots pronounced. Hindwing cream-coloured; outer margin with small ochreous brown spots forming more or less continuous line between Sc+R1, Rs, M1, M2, M3, CuA1 and CuA2; underside light ochreous, with marginal spots pronounced; fringes white.Male (n = 20) : Head whTympanal organs (n = 13): Transverse ridge more or less regularly rounded, medially more straight. Tympanic pocket extending slightly beyond transverse ridge. Tympanic drum ovoid, posteriorly not extended beyond transverse ridge. Tympanic bridge faintly sclerotized.Male genitalia (n = 13) , 27–29: Female (n = 7): Labial palpi: 1.6–2 mm; forewing length 12–16 mm; frenulum triple.Female genitalia (n = 7) : PapillaPageBreakThe species is known from Brazil in the Atlantic Forest  .Notes. The species was described from “one female collected in Brazil, near Rio de Janeiro”. Hence, the lectotype designated by a label by S. Bleszynski is not warranted. This designation is presumably based on the fact that Catharylla tenellus, or that there is indeed a deep divergence in the COI barcode between populations of this species.Specimens from Porto Seguro, Brazil show a divergence of 3.34% in COI barcode sequences with the specimen from Ubatuba, Brazil. In morphology, differences in male genitalia are also observed: in the specimens from Bertioga, Caraça, São Paulo and Ubatuba the costal arm of the valva is wide and 1/3 of the length of the cucullus, almost reaching its tip, and the dorsal edge at base is slightly produced . In the T. Léger & B. Landrysp. n.http://zoobank.org/7E0EB0BE-44C4-42EC-9F4D-2C923E9299E6http://species-id.net/wiki/Catharylla_coronataHolotype. ♂, with labels as follows: “Col. BECKER | 81552”; “BRASIL:ES | Linhares, 40m | 20-29.ii.1992 | V.O.Becker Col”; “HOLOTYPE | Catharylla | coronata | Léger & Landry” [red label]. Deposited in Becker Collection.Paratypes. 21 ♂, 4 ♀. BRAZIL: 5 ♂ with same data as holotype ; 2 ♂ with same data as holotype (1 used for DNA barcoding BC MTD 01891) except 05–09.iv.1992 (V. O. Becker n°82486); 6 ♂, 1 ♀ , Paranà, Rio Negro, 900 m, 8.ii.1973 (2 ♂), 10.ii.1973 (1 ♂), 11.ii.1973 (3 ♂), 13.ii.1973 (1 ♀) (A. & J. Razowski) (ISZP); 2 ♂, 2 ♀, Paranà, Curitiba, 920 m, 17.ii.1975 (1 ♂) (V. O. Becker n°10167), 20.ii.1975  (V. O. Becker n°10168), 12.iii.1975  (V. O. Becker n°10166), 10.x.1975 (1 ♂) (V. O. Becker n°4010) (Becker Coll.); 1 ♂ , Paranà, Castro, 950 m (E. D. Jones) (BMNH); 1 ♂, Paranà, Quatro Barras, 850 m, 27.ii.1970 (Laroca & Becker) (V. O. Becker n°15442) (Becker Coll.); 1 ♂  Rio de Janeiro, Novo Friburgo (BMNH); 1 ♂  Sao Paulo, 700 m (E. D. Jones) (BMNH); 1 ♂, 1 ♀ , Santa Catarina, Rio Vermelho, 968 m, 18.ii.1973 (♂), 28. ii. 1973 (♀) (A. & J. Razowski) (ISZP); 1 ♂, no locality data (V. O. Becker) (Becker Coll.).PageBreakCOI barcode sequence of paratype BC MTD 01890 (654 bp): ACTTTATATTTTATTTTTGGAATTTGAGCAGGAATAGTAGGAACATCATTAAGATTATTAATTCGAGCTGAATTAGGTAATCCTGGATCTCTTATTGGAGATGATCAAATCTATAATACTATTGTAACCGCTCATGCATTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGTGGATTTGGAAATTGATTAGTTCCCTTAATATTAGGAGCACCAGATATAGCTTTTCCTCGAATAAATAACATAAGATTTTGATTATTACCCCCCTCTTTAACTCTTTTAATTTCAAGAAGAATTGTAGAAAATGGAGCTGGAACAGGATGAACAGTTTACCCCCCACTTTCATCTAATATTGCCCATAGTGGAAGATCCGTAGATTTAGCAATCTTTTCCCTTCATTTAGCTGGAATTTCTTCAATTTTAGGAGCAATTAATTTTATTACAACAATTATTAATATACGAATCAATAATCTTTCATTTGATCAAATACCTCTTTTTGTTTGATCAGTAGGAATTACAGCTTTACTTCTTCTTTTATCATTACCAGTATTAGCTGGAGCTATTACTATACTTTTAACTGATCGAAATTTAAATACATCTTTTTTTGATCCCGCAGGAGGAGGAGATCCTATTTTATATCAACATTTACatharylla serrabonita and Catharylla tenellus, Catharylla coronata can be separated with characters of the male genitalia: the uncus is apically bifid and grooved on distal 1/5 in Catharylla coronata whereas it is only indented medially at apex in Catharylla serrabonita and Catharylla tenellus; the costal arm of the valva is short and the apex is curved inward in Catharylla coronata whereas the costal arm is longer and points postero-dorsally in the other two species; the transtilla forms a pair of sclerotized arms slightly bent inward distally, ventrally with a row of short spines increasing in size from base to apex whereas it forms a pair of short, narrow sclerotized arms with pointed tips, projecting posterad, and with a pair of brushes directed medio-ventrally in Catharylla tenellus and a pair of sclerotized arms strongly bent inward on distal 1/4 and with a string of long spines of same length medially along it in Catharylla serrabonita; the juxta is shorter than in Catharylla tenellus, and regularly narrowing toward apex whereas it is strongly narrowing on distal 1/4 in Catharylla serrabonita; the ventral projections of the juxta form a pair of shallow pockets whereas they are bell-shaped in Catharylla serrabonita and thumb-like in Catharylla tenellus; the vesica has a row of 6–7 cornuti in Catharylla coronata whereas it does not show any cornuti in Catharylla serrabonita and Catharylla tenellus. In the female genitalia of Catharylla coronata, the anterior angle of sternite VIII is not projected whereas it is rounded, projected anterad and covered with short spinules in Catharylla serrabonita, and projected downward in Catharylla tenellus. The anterior apophyses are quadrangular, anvil shaped whereas they are spine like in the other two species.From PageBreakline thin, light ochreous, apically faded; median transverse line light ochreous, concave on costal half, more or less disrupted; subterminal transverse line ochreous, curving toward base on costal half; R5 vein faintly marked apically with ochreous; outer margin ochreous with 7 pronounced dark brown spots more or less triangular between veins, sometimes connecting; fringes brass colored; underside white ochreous to ochreous, costal margin basally brown; outer margin with pronounced spots. Hindwing white to creamy white, usually with marginal brown spots between Sc+R1, Rs, M1, M2, M3, CuA1 and CuA2, forming more or less continuous line; fringes white; underside light ochreous, with dark brown marginal spots pronounced.Male (n = 21) : Head whTympanal organs (n = 7): Transverse ridge more or less regularly rounded. Tympanic pocket extending faintly beyond transverse ridge, rounded. Tympanic drum glomerular, not reaching transverse ridge.Male genitalia (n = 7) , 20: UncFemale (n = 4): Labial palpi: 1.6–2.2 mm long. Forewing length 14–16 mm. Frenulum triple.Female genitalia (n = 4) : PapillaThe species occurs in Brazil in the following states: Bahia, Espirito Santo, Paranà, Rio de Janeiro, Santa Catarina, Saõ Paulo .PageBreakcoronatus, a, um: crowned, referring to the longitudinal string of short spines of the transtilla in the male genitalia.The name comes from the latin Catharylla coronata is the sister species of the Catharylla tenellus + Catharylla serrabonita pair , Espírito Santo, Linhares, 40m, 25–30.i.1998 (V. O. Becker n°113929) ; 2 ♂, 1 ♀  with same except 20–29.ii.1992 (V. O. Becker n°81552) ; 2 ♂ with same data as holotype except 05–09.iv.1992 (V. O. Becker n°82486) (USNM); 10 ♂  Bahia, Camacan, Serra Bonita Reserve, 15°23'S, 39°33'W, 800 m, B. Landry, V. O. Becker, 1.iv.2011 (1 ♂), 2.iv.2011 (2 ♂), 3.iv.2011 , 5.iv.2011 (3 ♂), 6.iv.2011 (1 ♂), 7.iv.2011 (1 ♂) (MHNG); 1 ♂ with same data except vii.2010 (V. O. Becker) (Becker Coll.); 1 ♂  Bahia, Porto Seguro, A. d’Ajuda, 16°27'S, 39°03'W, 20 m, 12.vii.2009 (V. O. Becker n°144140) (Becker Coll.).COI barcode sequence of holotype LEP 979 (516 bp): TAGTTGGAACATCATTAAGACTATTAATTCGAGSAGAGTTAGGGAATCCTGGATCTCTTATTGGAGATGATCAAATTTATAATACTATTGKAACAGCTCATGSATTTATTATAATTTTTTTTATAGTTATACCAATTATAATTGGTGGATTTGGAAACTGACTAGTTCCATTAATATTAGGAGCCCCAGACATAGCTTTCCCCCGAATAAATAATATAAGATTTTGATTACTCCCCCCCTCTTTAACCCTTTTAATTTCCAGAAGAATTGTAGAGAATGGAGCTGGAACAGGATGAACGGTTTACCCCCCCCTTTCATCTAATATTGCTCATAGKGGAAGATCTGTAGATTTAGCAATTTTTTCTCTTCATTTAGSAGGAATTTCATCAATTTTAGGAGCAATTAATTTTATTACAACAATTATTAATATACGAATTAATAATTTATCTTTTGATCAAATACCGTTATTTGTCTGATCAGTTGGTATTACAGCTTTACTCCTTCTTTTATCTTTACPageBreakCatharylla coronata and Catharylla tenellus, Catharylla serrabonita can be separated by the zigzagging median transverse line with the short triangular dent at CuA2 and the pronounced creamy color of the hindwing. The male genitalia provide the best discriminant characters: in Catharylla serrabonita, the transtilla forms a pair of sclerotized arms bent inward in distal 1/4 and with a string of long spines of same length medially along it, whereas it forms a pair of short, narrow sclerotized arms with pointed tips projecting posterad, and with a pair of brushes directed medio-ventrally in Catharylla tenellus, and two sclerotized arms slightly bent inward distally, with a row of short spines increasing in size from base to apex in Catharylla coronata, and the juxta is apically narrow and pointed whereas it is triangular and regularly narrowed in Catharylla coronata and Catharylla tenellus. In female genitalia, the anterior angle of sternite VIII is projected anterad into a rounded protrusion covered with short spinules in Catharylla serrabonita, whereas it is projected downward in Catharylla tenellus and it is not projected in Catharylla coronata.From Male (n = 21) : Head whTympanal organs (n = 4): Transverse ridge more or less rounded, medially slightly flattened. Tympanic pocket extending faintly beyond transverse ridge, rounded. Tympanic drum glomerular, not reaching transverse ridge.Male genitalia (n = 4) –26: UncuFemale (n = 1): Labial palpi: 1.9 mm long. Forewing length: 14 mm. Frenulum triple.PageBreakbetween segment VIII and IX covered with microspines. Sternite VIII laterally about 5/3 length of tergite VIII; posterior margin of tergite VIII with line of setae; sternite VIII forming 2 triangular lobes regularly narrowing downward, not connected, densely covered with short spinules of same length; anterior angle of sternite VIII slightly projected anterad, rounded, covered with short spinules of same length. Anterior apophyses 0.03 × length of papillae anales. Sterigma membranous, covered with spinules. Ductus bursae about 3 × length of corpus bursae, narrow, basally directed downward and then bent upward. Corpus bursae elongate, ovoid, with one tiny signum.Female genitalia (n = 1) : PapillaThe species occurs in Brazil   & 46.The name comes from that of the Serra Bonita Reserve founded by Vitor O. Becker and Clemira de Souza. It is managed by Instituto Uiraçu in the State of Bahia, Brazil.Serra Bonita Reserve is located in the Atlantic Forest, in a hilly region of cacao plantations and scattered forest. Adults came late to light, usually after 23:00. Our molecular analysis of the COI barcode sequences highlighted that specimens from Serra Bonita respectively show 3.24 and 2.21 % base differences with those of Porto Seguro and Linhares. This divergence is possibly associated with slight morphological differences in male genitalia as shown in Diagnosis. We have not recovered any obvious synapomorphy for this group. It can be separated from the other Catharylla species based on the shorter forewing length, usually between 7.5 and 9.0 mm (maximum 10.5 mm). In male genitalia, the tegumen connection is more than two times longer than the uncus, the uncus is beak-shaped, with the apex narrowing to a point, the gnathos is bent at an angle of about 90°. In female genitalia, the basal line along the anal papillae is ventrally expanded onto a triangle, and the sterigma forms a pair of sclerotized pockets on each side of the middle, covered with short spines or spicules. The sterigma does not bear tiny setae on the ventral membrane of segment VIII.Notes. This group includes two species. The phylogenetic analyses restricted to the nuclear genes and the combined Bayesian analysis place the mayrabonillae group as sister to the chelicerata group , [Acre] Rio Branco, 1924 (Dengler) (SMNS); 2 ♀, Amazonas, Manaus, Reserva Ducke, AM-010, km 26, 2°55'S, 59°59'W, 15.xii.1993, U[ltra]V[iolet] Light (J. B. Sullivan & R. W. Hutchings) (USNM); 1 ♂ , Amazonas, Fonte Boa, ix.1906 (S. M. Klages) (BMNH); 1 ♀ , Federal District, Estaçao Florestal, Cabeca do Vedao, 1100m, 18.x.1971  (CNC); 2 ♂, Maranhão, Feira Nova, Faz[enda]. Retiro, 480m, 07°00'S, 46°26'W, 1–3.xii.2011 (V. O. Becker n°148263) (Becker Coll.); 2 ♀, Pará, Belém, 20m, i.1984 (V. O. Becker n°46981) (Becker Coll.); 1 ♀, Pará, Capitao Poco, 25–31.i.1984 (V. O. Becker n°97880) (Becker Coll.); 1 ♂, Rondonia, Cacaulãndia, 140m, xi.1991 (V. O. Becker n°79592) (Becker Coll.); 1 ♀, Rondonia, 62km S[outh] Ariquemes, Fazenda Rancho Grande, 165m, 10°32'S, 62°48'W, 18–26.iv.1991 (R. Leuschner) (USNM). COLOMBIA: 1 ♂, Valle, J[un]ct[ion]. Old B’[uena]v[en]tura R[oa]d. and Rio Dagua, 50m, 8.ii.1989 (J. B. Sullivan) (USNM). COSTA RICA: 1 ♀ , Alajuela, Area de Conservacion Guanacaste, Estacion Caribe, 12.xi.2007 (S. Rios & H. Cambronero) (INBio); 1 ♀, Alajuela, Area de Conservacion Guanacaste, Rio Negro, 25.i.2009 (H. Cambronero & F. Quesada) (INBio); 2 ♂ , Alajuela, San Carlos, Arenal National Park, Send[ero] Pilón, Rio Celeste, 700m, light trap, 17–19.x.2001 (G. Rodriguez) (INBio); 1 ♂, Prov[incia] Guanacaste, F[in]ca Pasmompa, Est[acion] Pitilla, 5km SO S[an]ta Cecilia, 400m, xii.1990 (P. Rios & C. Moraga) (INBio). ECUADOR: 4 ♀ with same data and deposition as holotype; 4 ♂, 8 ♀ (1 ♂ used for DNA barcoding BC MTD 01844) with same data (USNM); 1 ♀ , Napo, 6km NW Tena, Lumu Caspi, 0°54'37"S, 77°49'32"W, 590m, 29.ix.2002 (Schouten Coll.); 1 ♀ , Pastaza, 1km N Santa Clara, 1°16'02"S, 77°52'57"W, 630m, 28.ix.2002 (Schouten Coll.). FRENCH GUIANA: 1 ♂, 4 ♀ , Saint Jean de Maroni (E. Le Moult) (BMNH); 2 ♀, Piste Nancibo, km 6, 4°41'N,; 52°25'W, in logged rain forest, at 125W[atts] mer[cury]-vapor light and 15W[atts] U[ltra]V[iolet], 11.i.1985 (J.[-]F. Landry) (USNM); 1 ♂, Roura, Montagne des Chevaux, xii.2008 (S. Delmas) (MHNG); 1 ♂ , Cayen[ne] (BMNH). GUYANA: 1 ♀ , Omai, vi.1908 (S. M. Klages) (BMNH); 1 ♀ , Potaro i.1908 (S. M. Klages) (BMNH). PANAMA: 1 ♀, Rio Trinidad, 12.iii [no year data] (A. Busck) (USNM). PERU: 1 ♂ , Agnaytia, Huallaga, 400m, ix.1961  (CNC); 1 ♀ , Yurimaguas, PageBreakHuallaga 14.iv.[19]20 (CMNH). SURINAME: 2 ♀ , Kabo, 5°16'N, 55°44'W, Saramaca, black light, respectively 15–16.iii.1983 and 13–14.i.1983 (K.E.Neerling) (Schouten Coll.); 1 ♀ , Sipaliwini Distr[ict]., Tibiti area, Kabo Creek, partly swampy primary forest on hilly slopes, ca 2km from river, vi.1989 (J. Beerlink) (Schouten Coll.).Other specimen examined. 1♀ (used for DNA sequencing Lep 1126), Peru, Huánuco, Rio Llullapichis, Panguana, 74,945°W / 9,614°S, 23.9.–10.10.2011 (SMTD).COI barcode sequence of paratype 07-SRNP-113921 (654 bp): ACATTATATTTTATTTTCGGGATTTGAGCAGGTATAGTAGGAACTTCACTTAGATTATTAATTCGTGCTGAATTAGGTAACCCTGGCTCTCTTATTGGAGATGATCAAATTTATAATACTATTGTAACAGCCCATGCATTTATTATAATTTTTTTTATAGTTATACCTATTATAATCGGTGGATTTGGAAATTGATTAGTTCCTTTAATATTAGGGGCACCAGATATAGCTTTCCCTCGAATAAATAACATAAGATTTTGATTATTACCACCATCATTAACTCTTTTAATTTCTAGAAGAATTGTAGAAAATGGAGCTGGAACAGGATGAACAGTTTATCCACCTTTATCATCTAATATTGCCCATGGGGGTAGATCTGTAGATTTAACAATTTTTTCATTACATTTAGCTGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACAACAATTATTAATATACGAATTAATAATTTATCATTTGATCAATTATCATTATTTATTTGATCAGTAGGAATTACTGCTTTACTTTTATTATTATCATTACCAGTTTTAGCTGGGGCTATTACTATACTTTTAACTGATCGAAATCTTAATACATCATTTTTTGATCCAGCAGGAGGAGGAGATCCAATTTTATATCAACATTTAmayrabonillae group are the shape of the forewing outer margin, which is slightly produced apically in Catharylla mayrabonillae and not produced in Catharylla paulella, and the forewing median transverse line with two strongly pronounced spots at 1/3 and 2/3 in Catharylla paulella, whereas these spots are lacking in Catharylla mayrabonillae. The hindwing of Catharylla mayrabonillae has a faded subterminal transverse line on costal half whereas the hindwing of Catharylla paulella lacks this marking. In male genitalia, the heavily sclerotized sacculus bears a dorso-lateral sclerotized string of short spines on distal 1/4 whereas the two processes of the costa are S-shaped in Catharylla paulella, and the apex of the phallus is trifid, rounded medially, shortly triangular laterally, whereas it is simply rounded in Catharylla paulella. In female genitalia, the sterigma forms double rounded cavities with a mustachio-shape arrangement of short spines in ventral view, and the ductus bursae is wide, progressively widening toward corpus in Catharylla mayrabonillae, whereas the sterigma forms a pair of shallow rounded pockets on each side of middle and the ductus bursae is narrow, with the rounded corpus bursae clearly differentiated from it in Catharylla paulella.The best discriminant characters externally between the two species of the PageBreakdorsally ashen brown, tibia and tarsomeres ochreous, distally ringed with dark brown. Midleg femur white, tibia light ochreous, basally brown, tarsomeres II–V ochreous with tips ringed white. Hindleg white, except tarsomeres, as in midleg. Abdomen dull white. Forewing length: 7.5–8.5 mm; with apex slightly produced; costal line thin, ochreous or white in basal half, white in apical half; median transverse line ochreous, slightly undulated; subterminal transverse line ochreous; transverse lines enlarging into brown spot on costal margin with ochreous bar on costa following subterminal transverse line; terminal sector with light ochreous between veins, margin with thin, dark brown line from apex to CuA1, with two dark brown spots in cubital sector, with spot between CuA1 and CuA2 slightly displaced toward base; fringes brass colored; underside light ochreous with some brownish scales, with thin brown margin. Hindwing white with thin transverse subterminal line faded ochreous, in continuity with forewing median transverse line; outer margin line pronounced, dark brown; underside dull white with thin faded brown margin; fringes white.Male (n = 17) : Head win=7): Transverse ridge regularly rounded, medially slightly flattened. Tympanic pockets broadly rounded, extended widely beyond transverse ridge, connected medially at base of praecinctorium. Tympanic drum bean-shaped, elongated, extended beyond tympanic pockets.Tympanal organs  , 31: UncFemale (n = 37): Labial palpi length: 1.1–1.3 mm. Forewing length: 9.5–10.5 mm; frenulum triple.PageBreakside of enlargement and posterior section. Corpus bursae circular to elongate, about as long as tergite VII; single signum faintly pronounced.Female genitalia (n = 16) : PapillaCatharylla and the only one so far found in Central America and in Venezuela, Columbia, Ecuador and Peru.The species has been found so far in Panama, Costa Rica, Colombia, Venezuela, Guyana, Suriname, French Guiana, Ecuador, Peru and Brazil  . It is tCatharylla mayrabonillae is named in honor of Ms. Mayra Bonilla of San Jose, Costa Rica, in recognition of her artistic portrayal of the biodiversity and ecosystems of Costa Rica and her many years of support for the existence of the rain forest in Area de Conservacion Guanacaste.The relatively strong COI barcode divergence of 4.34% between samples LEP 1126 from Peru and 07-SRNP-113921 from Costa Rica  is notabSchaus, 1922http://species-id.net/wiki/Catharylla_paulellaCatharylla paulella Schaus, 1922: 131; Holotype. ♀, with labels as follows: “Sao Paulo | S.E. Brazil.”; “Collection | W[illia]mSchaus”; “Type No. | 25533 | U.S.N.M.” [orange label]; “SLIDE | SB ♀ | No.4641” [light blue label]; “Catharylla | paulella | type Sch[au]s” [hand written]; “Genitalia Slide | By SB | USNM 111,535” [green rectangular label with thin black line submarginally]. Deposited in USNM.Other specimens examined. 2 ♂, 7 ♀. BOLIVIA: 2 ♂ , Prov.[incia] del Sara, 450 m, iv.1910 (J. Steinbach) . BRAZIL: 1 ♀ , Federal District, Planaltina, 15°35'S, 47°42'W, 1000 m, 3.xi.1977 (V. O. Becker n°22055) (Becker Coll.), 1 ♀  with same locality, 16.x.1990 (V. O. Becker n°96854) (Becker Coll.); 1 ♀, Maranhão, Feira Nova, Faz[enda]. Retiro, 480m, 07°00'S, 46°26'W, 1–3.xii.2011 (V. O. Becker n°148263) (Becker Coll.); 1 ♀ , Mato Grosso, Urucum, 15 miles S[outh]. of Columbá, 650 f[ee]t, 19. iv. [19]27, at light (C. L. Collenette) (BMNH); 1 ♀, Parà, Belém, 20m, i.1984 (V. O. Becker n°46993) (Becker Coll.); 1 ♀ , São Paulo (BMNH); 1 ♀ , São Paulo, São Luiz do Paraitinga, 23°20'S, 45°06'W, 900 m, 13–20.iii.2001 (V. O. Becker n°132357) (Becker Coll.).PageBreakTATTAATTCGTGCTGAATTAGGTAATCCTGGATCTCTTATTGGTGATGATCAAATTTATAATACTATTGTAACAGCTCATGCATTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGTGGATTTGGAAATTGATTAGTTCCTTTAATATTAGGTGCACCAGATATAGCTTTCCCTCGAATAAATAATATGAGATTTTGATTATTACCCCCATCATTAACTCTTTTATTTT?TAGAAGAATTGTCGAAAATGGAACTGGAACAGGATGAACAGTTTACCCACCCTTATCATCCAATATTGCTCATAGAGGTAGATCAGTAGATCTAGCAATTTTTTCTTTACATTTGGCTGGAATTTCATCAATCTTAGGAGCTATTAATTTTATTACAACAATTATCAATATACGAATTAATAATTTATCTTTTGATCAATTATCATTATTTATTTGATCTGTAGGTATTACAGCTTTACTTTTATTATTATCATTACCAGTTCTAGCTGGAGCTATTACTATACTTTTAACTGATCGAAATCTTAATACATCATTTTTTGATCCTGCAGGAGGAGGTGATCCTATCTTGTATCAACATTTACOI barcode sequence of specimen LEP 965 (654 bp): ACATTATATTTTATTTTTGGAATTTGAGCAGGTATACTAGGAACTTCACTTAGATCatharylla species by the forewing median transverse line with two strongly pronounced spots at 1/3 and 2/3. The forewing is also sparkled with dark brown scales, which is unique in the genus. In male genitalia, the two S-like projections of the costal arm of the valva discriminate this species from the other species of Catharylla. In female genitalia, the sterigma forms a pair of shallow rounded pockets on each side of middle, and the ductus bursae is narrow, with the rounded corpus bursae clearly differentiated from it in Catharylla paulella, whereas it forms double rounded cavities with a mustachio-shape arrangement of short spines in ventral view, and the ductus bursae is wide, progressively widening toward corpus in Catharylla mayrabonillae.This species can be easily separated from the other Male (n = 2): Head with ochreous chaetosemata. Antenna ochreous with white scales, with patch of dark brown scales at base. Maxillary palpus light ochreous, white tipped. Labial palpus: 1.4–1.7 mm long, light ochreous, white tipped. Thorax light ochreous at collar. Foreleg coxa whitish ochreous, femur light ochreous, dorsally ochreous; tibia and tarsomeres greyish brown, distally ringed with dark brown. Midleg and hindleg whitish ochreous; midleg tibia basally brown, hindleg tibia white; midleg and hindleg tarsomeres with white tips. Forewing length: 7–8 mm; costal line thin, brown or dirty white; median transverse line ochreous, with two dark brown strongly pronounced spots at 1/3 and 2/3; subterminal transverse line thin, ochreous, with small triangular spot on costal margin; with ochreous bar on costal margin following subterminal transverse line; outer margin ochreous with short dark brown lunules or dashes; fringes brass colored; underside light ochreous with brownish suffusion; with pronounced marginal spots. Hindwing white; outer margin with thin ochreous line in apical half; fringes white; underside dull white with dark brown marginal spots more or less connected on apical half.PageBreaklus with apex more thickly sclerotized, with blunt apical margin, with short triangular ventral projection; vesica covered on basal 1/4 with tiny spicules, with barely visible microspicules all along, with one wide and curved cornutus at about 1/4 length of phallus.Male genitalia (n = 2) , 33: UncFemale (n = 7) , 10: LabTympanal organs (n = 5) : TransveFemale genitalia (n = 5) : PapillaThe species has been found in Brazil  and in Bolivia .Catharylla hibisca to specimens that appear to be Catharylla paulella. The BMNH São Paulo specimen is associated with slide n° 17692, but the genitalia on this slide seem to be wrongly associated, given the inscription “wrong abdomen?” on the label, as well as the size of the abdomen, which is much bigger than those of Catharylla paulella. Therefore, this specimen cannot be identified with certainty. An error is possible in the association of the sexes of this species as there are no series of both sexes from the same locality or other means of associating them with 100% confidence.The original description doesn’t mention the original number or sex of the specimens but it is assumed that there was only one. S. Bleszynski gave the new name of The number of bases obtained for each barcode sequence is given in Catharylla species. We observe a relatively high divergence between different barcode haplotypes in Catharylla bijuga (5.05%), Catharylla mayrabonillae (4.34%), Catharylla serrabonita (3.24%) and Catharylla tenellus (3.34%), sometimes possibly associated with differences in morphological characters  and with geographical distances. The divergence between Catharylla chelicerata samples LEP 1703 and LEP 1704 is of 0.15% (1 base) because they are issued from the same population. Sample LEP 1290 differs from samples LEP 1703 and LEP 1704 respectively by 0.62 and 0.46%. We observe no variation between samples LEP 1708, LEP 1709, LEP 1710 and LEP 1888 because they are all issued from the same population. The inter-specific variation in COI barcode sequences (6.29–16.84%) is always higher than the intraspecific variation (0.15–5.05%).As most of the data were restricted to few sequence samples per species, and some of the sequences came from the same populations, we don’t have the definitive picture of the intraspecific variation in the COI barcode sequences of The 21 analysed characters are listed in PageBreakPageBreakPageBreakCatharylla is supported by the analysis of mol_1 and the combined Bayesian analysis  and by the analyses of nucl_1 and nucl_2 . Three synapomorphies  and one non-unique apomorphy  support the group. The mayrabonillae group is well supported in all analyses  except in the morphology-based analysis (BS support = 60), because no clear synapomorphy was found for the group. However, two non-unique synapomorphies and one reversal (observed only once in Catharylla) are observed . The tenellus group is well supported by the morphology-based analysis (BS support = 97) with four clear synapomorphies  and two reversals , but show no support in other analyses, probably because only the barcode sequence was available for Catharylla coronata. The closer relationship between Catharylla serrabonita and Catharylla tenellus within the tenellus group is supported by the combined Bayesian analysis and by one non-unique apomorphy (but unique in Catharylla). The tenellus group as represented in the analyses of nucl_1 and nucl_2 (Catharylla serrabonita + Catharylla tenellus) is well supported . The chelicerata group is well supported by the morphology-based analysis (BS support = 96) and the combined Bayesian analysis (PP = 1.00). The group shows two synapomorphies , one non-unique apomorphy (7:1) and one reversal (11:0). Unfortunately, no sequence was available for Catharylla gigantea, thus we cannot compare the morphology with the molecular-based analyses. The closer relationships between the chelicerata and the mayrabonillae groups is strongly supported by the nucl_1 analysis (BS support = 100) and the combined Bayesian analysis (PP = 1.00). Two reversals  support the group. The position of Catharylla bijuga as sister group of the other Catharylla species is weakly supported by the combined Bayesian analysis (PP = 0.90), and show no support in other analyses. The position of Micrelephas pictellus as sister group of Catharylla is supported by the analysis of mol_2 and nucl_2 (respective BS supports of 91 and 100). This node is supported by one synapomorphy . Node 10 (Argyria lacteella+ Micrelephas pictellus+ Catharylla) is supported by the analysis of mol_1 (BS support = 100) and the combined Bayesian analysis (PP = 1.00). One synapomorphy (2:1) supports the group. The monophyly of the two Crambus species chosen as outgroups is well supported in the mol_1 analysis (BS support of 100). The settings of MrBayes do not allow to choose two outgroups, therefore the monophyly of the two Crambus species is not supported by the combined Bayesian analysis. Three non-unique synapomorphies support the genus based on these two species . The synapomorphies of the different groups highlighted by the phylogeny are reported here below:The monophyly of Chelicerata group (node 3)8:1. Apex of valva quadriangular, truncated9:2. Gnathos regularly curvedTenellus group (node 5)5:1. Presence of a dorsal furrow on the uncus7:3. Apex of uncus slightly bifid10:1. Transtilla present20:1. Postvaginal sterigma absentCatharylla (node 8)11:1. Lateroventral projections on juxta17:1. Posterior apophyses/papillae anales < 0.519:1. Ventral membrane of segment VIII with tiny setaeCatharylla + Micrelephas (node 9)6:1. Uncus dorsally bare, or with few setaeCatharylla + Micrelephas + Argyria (node 10)2:1. Length of labial palpi/eye diameter < 2/1PageBreakPageBreakPageBreakCatharylla occurs northward from Middle America with locality records in Costa Rica (Area de Conservacion Guanacaste) at a latitude of 10°54 N, southward to Rio Vermelho  at a latitude of 27°30 S, from sea level up to 1300 m . Within Catharylla, species and species groups show distinct distribution patterns. The chelicerata group is widespread in the Northern Amazonian rainforest of Brazil, and in the three Guyanas , whereas Catharylla paulella is widespread from Feira Nova (07°00 S) down to São Paulo (23°35 S)  , in the Chaco formation   while haplotype BC MTD 1840 is from the Humid Guyana province . Catharylla mayrabonillae barcode haplotype 07-SRNP-113921 is found in the Carribean subregion, whereas haplotype LEP 1126 is found in the Amazonian subregion. The barcode haplotypes of the Catharylla serrabonita populations from the two coastal localities of Linhares and Porto Seguro are more closely related, whereas the haplotype from the population of the forested hills of the Serra Bonita Reserve clearly diverges from the other two  . They arFederal) , 44, wheFederal) . CatharyBolivia) , 44, whiBolivia) . Catharydiagonal  formed bdiagonal , constitther two .PageBreakCatharylla (nodes 7 & 8). The reduced number of taxa , as well as the quality of the datasets (complete sets of genes for nucl_1 and nucl_2 vs partly complete sets of genes in mol_1 and mol_2) explain the better results obtained in nucl_1 and nucl_2, because taxa with incomplete data tend to lower the resolution and the bootstrap supports of the tree  because of the great divergence of these sequences from those of other Catharylla species. However, the clear support brought by the morphological analysis places Catharylla coronata together with Catharylla serrabonita and Catharylla tenellus. The neighbor-joining (NJ) analysis of barcode sequences of Catharylla species (not represented here) places Catharylla coronata together with Catharylla serrabonita (divergence of 7.59% with the sequence BC MTD 1843) and Catharylla tenellus (divergence of 7.3% with the sequence BC MTD 1842), and thus corroborates the findings of the morphology-based analysis. Moreover, the species is morphologically very similar to Catharylla serrabonita especially regarding the transtilla in the male genitalia. The basal position of Catharylla bijuga in Catharylla, as sister taxon of other Catharylla species, is doubtful (PP = 0.90) and may be an artefact due to the great divergence of the barcode sequence (the lowest divergence with other Catharylla species is of 9.7% with sample BC MTD 1843 of Catharylla serrabonita). The supports brought by the combined Bayesian analysis have to be carefully considered since the results are sensitive to small variations of the taxon and character sampling, and the posterior probabilities tend to overestimate the strongness of the nodes  along with Argyria, Urola and Vaxi, and Micrelephas in Crambini , Catharylla mayrabonillae (4.34%), Catharylla serrabonita (3.24%) and Catharylla tenellus (3.34%) could therefore suggest that these different barcode haplotypes represent different species. Some morphological variation in male genitalia was found in Catharylla serrabonita and Catharylla tenellus , as well as Catharylla serrabonita and Catharylla tenellus (both collected in Porto Seguro), while Catharylla coronata and Catharylla serrabonita are vicariant, Catharylla serrabonita being distributed north of Linhares, whereas Catharylla coronata is found south of this locality. The patterns of distribution suggest that the diversification of the tenellus group might have occurred in the Atlantic Forest, or have occurred in a wider area that reduced afterwards. In Catharylla tenellus, two forms of male valva linked to different COI barcode sequences seem to be geographically separated, with the form from Ubatuba (associated to barcode sequence BC MTD 1842), Bertioga, São Paulo (São Paulo State), and Caraça (Minas Gerais) occurring more to the south than the form from Porto Seguro . The third form, observed in Caraça (Minas Gerais) suggests that this locality could represent a point of contact between the two other forms. Regarding Catharylla serrabonita, the locality of Serra Bonita is a moist forest of middle elevation (800 m)  , while t


Drosophila SCA3 models with Hsp104, a powerful protein disaggregase from yeast, which is bafflingly absent from metazoa. Hsp104 suppressed eye degeneration caused by a C-terminal ataxin-3 (MJD) fragment containing the pathogenic expanded PolyQ tract, but unexpectedly enhanced aggregation and toxicity of full-length pathogenic MJD. Hsp104 suppressed toxicity of MJD variants lacking a portion of the N-terminal deubiquitylase domain and full-length MJD variants unable to engage polyubiquitin, indicating that MJD-ubiquitin interactions hinder protective Hsp104 modalities. Importantly, in staging experiments, Hsp104 suppressed toxicity of a C-terminal MJD fragment when expressed after the onset of PolyQ-induced degeneration, whereas Hsp70 was ineffective. Thus, we establish the first disaggregase or chaperone treatment administered after the onset of pathogenic protein-induced degeneration that mitigates disease progression.There are no effective therapeutics that antagonize or reverse the protein-misfolding events underpinning polyglutamine (PolyQ) disorders, including Spinocerebellar Ataxia Type-3 (SCA3). Here, we augment the proteostasis network of  Drosophila models of SCA3. Hsp104 has no homologue in animals, but has an unusual ability to dissolve PolyQ aggregates in vitro, an activity that could be harnessed therapeutically. Indeed, Hsp104 suppressed degeneration caused by a C-terminal ataxin-3 (MJD) fragment containing the pathogenic expanded PolyQ tract, which accumulates in disease. However, Hsp104 enhanced aggregation and toxicity of full-length pathogenic MJD. Hsp104 rescued forms of MJD unable to engage polyubiquitin or with a deletion in the deubiquitylase domain indicating that MJD-ubiquitin interactions hinder protective Hsp104 activities. Importantly, Hsp104 suppressed toxicity of a C-terminal MJD fragment when expressed after the onset of PolyQ-induced degeneration, whereas Hsp70 was ineffective. Thus, we establish the first disaggregase or chaperone treatment administered after the onset of pathogenic protein-induced degeneration that mitigates disease progression.There are no effective therapeutics for any of the neurodegenerative disorders caused by expanded polyglutamine (PolyQ) tracts including Spinocerebellar Ataxia Type-3 (SCA3). These disorders are connected with the misfolding and aggregation of proteins bearing expanded PolyQ tracts in the neurons of affected individuals. In SCA3, ataxin-3 (MJD) is the protein that bears the PolyQ expansion and forms insoluble aggregates. Here, as a therapeutic strategy we introduce Hsp104, a powerful protein disaggregase from yeast, into  Many neurodegenerative diseases, such as Alzheimer's Disease, Parkinson's Disease (PD), prion disease, and the collection of polyglutamine (PolyQ) disorders, including Huntington's Disease (HD) and the Spinal Cerebellar Ataxias (SCAs), are characterized by the formation of protein inclusions in the nervous system Despite the extraordinary structural stability of amyloid, a protein disaggregase from yeast, Hsp104, can rapidly solubilize amyloid. Hsp104 is a hexameric AAA+ (ATPases Associated with diverse cellular Activities) protein that couples ATP hydrolysis to translocation of substrate through a central pore, thus prying individual monomers from the amyloid fiber Curiously, Hsp104 has no homologue in metazoa. Indeed, until recently it was unclear whether the metazoan proteostasis network possessed any coupled protein disaggregase and reactivation machinery. It is now clear that Hsp110, Hsp70, and Hsp40 collaborate to promote the dissolution and reactivation of disordered aggregates in vitro, with the Josephin domain forming SDS-soluble linear polymers that then convert into SDS-insoluble PolyQ-driven amyloid fibers Spinocerebellar Ataxia Type 3 or Machado-Joseph Disease (MJD/SCA3) is the most prevalent dominantly inherited ataxia C. elegans, Hsp104 prevented aggregation and toxicity of GFP-tagged PolyQ in vivo after aggregates have already formed and degeneration has begun; a situation likely to mimic an actual therapy. Therefore, we created novel Hsp104 Drosophila lines to exploit well-characterized models of disease in combination with powerful genetic tools to temporally control the expression of Hsp104 after disease-associated aggregation and degeneration has begun.Hsp104 has been introduced to combat protein-aggregation disease in metazoan systems with various levels of success in vivo.Our studies reveal surprisingly distinct interactions of Hsp104 with the full-length versus a truncated version of the MJD protein. Importantly, we establish that Hsp104 possesses the ability to suppress the progression of degeneration when activated subsequent to onset of expression of the disease protein. These data indicate that protein context is central in Hsp104 interactions, and that Hsp104 displays the ability to halt the progression of pre-established disease Drosophila to evaluate its ability to prevent and potentially reverse aggregation of disease-associated human proteins, readily available in various fly models of disease. To achieve strong expression of the Hsp104 protein in the fruit fly, we codon-optimized the transgene for Drosophila (see ACAAA) before the start codon Drosophila using the GAL4/UAS system glass gene element for driving GAL4 expression, we instead used a driver line with reduced expression  and the truncated C-terminal region of the protein containing the expanded glutamine tract (MJDtrQ78) To probe the mechanism underlying the dichotomous results found for the Hsp104 interaction with MJDtrQ78 and MJDnQ78, an in-depth investigation of the protein aggregates was performed. To slow protein aggregation such that we could analyze underlying protein accumulations in detail, we expressed the transgenes in the eye with an adult-onset driver rhodopsin1(rh1)-GAL4. Analysis of the PolyQ protein accumulations showed that Hsp104 altered the kinetics of inclusion formation for both MJD protein isoforms. By cryosectioning and subsequent immunohistochemistry (IHC), MJDtrQ78 formed compact inclusions that increased in size over time , top rowTo examine protein accumulation by biochemical methods, we used SDD-AGE (Semi-Denaturing Detergent–Agarose Gel Electrophoresis), a protein agarose gel technique that can resolve amyloid aggregates in vitroNext, we assessed MJDnQ78 misfolding. In contrast to the truncated MJDtrQ78 isoform, the pathogenic full-length MJDnQ78 initially formed amorphous inclusions that did not become more numerous after day 1 . Thus, tWhile it is known that in select conditions, Hsp104 promotes amyloid formation of specific yeast prions We also examined variants lacking DUB activity through mutation of the active site in the Josephin domain, MJD-Q88-C14A , which cTo uncover additional mechanistic insight into the interactions with Hsp104, we examined inclusion formation and kinetics with adult-onset rh1-GAL4 expression. By IHC, the MJD variants formed accumulations in a manner roughly consistent with severity of eye degeneration , precludA summary of the effect of Hsp104 on MJD variants is presented in DPLDWB), which is structurally identical to wild-type but functionally inactive DPLDWB were innocuous  into Hsp104 to ensure that Hsp104 could not engage substrate or hydrolyze ATP, creating the mutant known as Double Pore Loop Double Walker B  the toxic MJDtrQ78 protein driven directly by a gmr element such that the disease-associated protein was constitutively expressed in the eye; (2) a drug-inducible gmr-GAL4 driver known as “GeneSwitch” (gmr-GS) to activate GAL4 expression only in the presence of the drug RU486 (mifepristone) Hsp104 is unique in its capacity to reverse pre-existing amyloids in yeast and DPLDWB had no effect  and progressed in severity to d7 , Fig. 10o effect . Hsp104 Next, we examined the underlying protein aggregates by SDD-AGE and Western immunoblot. We observed that gmr-MJDtrQ78 had high levels of amyloid, and this was lessened with time  . When tuin vivo, which is its ability to mitigate the course of protein-aggregation disease even after it has already initiated. Indeed, our studies show that Hsp104 is able to mitigate disease progression once it has begun, unlike the classical metazoan chaperone Hsp70. These studies provide new insight into the in vivo effects of Hsp104 in the context of a therapeutic agent.Here, we reveal key novel insights into the efficacy and interactions of Hsp104 with the pathogenic PolyQ protein MJD. Our studies reveal the surprising finding that Hsp104 interacts differentially with different forms of the MJD protein. Hsp104 is a potent suppressor of toxicity of the truncated protein, but an enhancer of toxicity of the full-length protein. These differences are determined by specific domains of MJD that are not directly implicated in aggregation. Our findings have also uncovered a heretofore unrecognized and important application of Hsp104 Our detailed investigations of the effects of Hsp104 on the MJD protein led to the unexpected result that Hsp104 has opposite effects on the toxicity of different versions of the MJD protein (MJDtrQ78 and MJDnQ78), despite the fact that these proteins contain the same pathogenic PolyQ stretch. These disparate actions indicate that Hsp104 might be a useful probe to understand the nature of aggregates and the toxicity imposed by them. Hsp70 suppressed both MJDtrQ78 aggregation and toxicity , 3C. By What might these biochemical changes be? Hsp104 can disrupt toxic soluble oligomers of various proteins, including Sup35, which may help explain why Sup35 prion formation is not intrinsically toxic to yeast In other settings, it has been suggested that chaperone-initiated formation of large, insoluble amyloid aggregates can actually be protective by sequestering potentially toxic pre-fibrillar conformers Moreover, our findings demonstrate that an agent with a mitigating effect on the truncated version of the MJD protein may act in a different manner against the full-length MJD protein. Thus, what is good for one may not be beneficial for the other. In MJD/SCA3, as well as other neurodegenerative diseases, fragmentation of the disease protein may initiate aggregation and this process is critical for disease progression Although Hsp104 enhances MJDnQ78 amyloidogenesis and toxicity, we found that elimination of functional domains not implicated in PolyQ aggregation facilitated the ability of Hsp104 to suppress MJD-associated degeneration. Elimination of UIM functionality or removal of a component of the Josephin domain (exon 2) restored the remodeling capacity of Hsp104. This suggests that MJDnQ78 pathogenicity is not intrinsically intractable, but is capable of being suppressed by Hsp104 if other domains of the protein are inactivated . Alternatively, potentiated or MJDnQ78-optimized Hsp104 variants might be developed that are able to overcome these hindrances via increased unfolding power Our studies underscore the importance of protein context in studying protein-misfolding diseases. Within the protein itself, neighboring domains not thought to be involved in aggregation may be impacting accumulation kinetics and the biochemical properties of inclusions in vitro have characterized aggregation of the full-length, pathogenic MJD protein as a two-step process in which the protein assembles first into SDS-soluble fibrillar polymers associating via the Josephin domain, and then converts to SDS-insoluble amyloid fibers driven by the PolyQ domain in vivo as well. Indeed, it is consistent with our observation that full-length MJDnQ78 forms amorphous accumulations that appear visually by IHC before they can be observed as SDS-insoluble amyloid aggregates by SDD-AGE  may increase efficiency of such interactions and enable Hsp104 to rescue disease phenotypes. Moreover, if UIM binding to poly-ub chains is impairing access of Hsp104 to MJD, this suggests that co-administering an agent to modulate function of a neighboring domain may affect the access of a treatment to the aggregation-prone domain. Indeed, increasing global DUB activity coupled with Hsp104 induction could overcome antagonism due to poly-ub chains.DrosophilaChaperone treatment, and examination of Hsp70 in particular, has been an exciting avenue of research in the battle to combat and contain neurodegenerative disease An inducible system is particularly well suited for Hsp104 because of its unique ability to rapidly dismantle pre-existing amyloid aggregates. Since metazoan chaperones can only very slowly depolymerize amyloid Our experimental paradigm offers the exciting possibility to address the efficacy of Hsp104 (or other molecules) in a more genuine therapeutic setting. Indeed, we found that turning on Hsp104 was able to significantly suppress disease-associated degeneration. Interestingly, however, Hsp104 did not disaggregate MJDtrQ78 amyloid in these experiments . Thus, Hin vitro and in the most appropriate animal models. Moreover, the fact that Hsp104 is well tolerated by mammalian systems is encouraging Naturally, several barriers must be surmounted to translate Hsp104 into a therapeutic agent for human neurodegenerative disease Clostridium botulinum, as a therapeutic agent. Despite being a deadly toxin, botulinum toxin variants have found key clinical applications due to their highly potent and selective ability to cleave SNARE proteins and prevent secretion Finally, the concept of using a yeast protein as the basis for a therapeutic agent might at first glance seem implausible. However, it must also have seemed equally implausible to use a lethal protein toxin from the bacterium, DPLDWB were generated by standard techniques using the pUAST vector Drosophila and a Kozak sequence (ACAAA) was added prior to the start codon Transgenic flies expressing UAS-Hsp104 and UAS-Hsp104The full sequence of codon-optimized Hsp104 is:ATGAACGATCAGACCCAGTTCACCGAGCGCGCCCTGACCATCCTGACCCTGGCCCAGAAGCTGGCCAGCGATCACCAGCACCCCCAGCTGCAGCCCATCCACATCCTGGCCGCCTTCATCGAGACCCCCGAGGATGGCAGCGTGCCCTACCTGCAGAACCTGATCGAGAAGGGCCGCTACGATTACGATCTGTTCAAGAAGGTGGTGAACCGCAACCTGGTGCGCATCCCCCAGCAGCAGCCAGCCCCAGCCGAGATCACCCCAAGCTACGCCCTGGGCAAGGTGCTGCAGGATGCCGCCAAGATCCAGAAGCAGCAGAAGGATAGCTTCATCGCCCAGGATCACATCCTGTTCGCCCTGTTCAACGATAGCAGCATCCAGCAAATCTTCAAGGAGGCCCAGGTGGATATCGAGGCCATCAAGCAGCAGGCCCTGGAGCTGCGCGGAAACACCCGCATCGATAGCCGCGGAGCCGATACCAACACCCCCCTGGAGTACCTGAGCAAGTACGCCATCGATATGACCGAGCAGGCCCGCCAGGGAAAGCTGGACCCAGTGATCGGACGCGAGGAGGAGATCCGCAGCACCATCCGCGTGCTGGCCCGCCGCATCAAGAGCAACCCATGCCTGATCGGAGAGCCAGGAATCGGCAAGACCGCCATCATCGAGGGAGTGGCCCAGCGCATCATCGATGATGATGTGCCAACCATCCTGCAGGGAGCCAAGCTGTTCAGCCTGGATCTGGCCGCCCTGACCGCCGGCGCCAAGTACAAGGGCGATTTCGAGGAGCGCTTCAAGGGCGTGCTGAAGGAGATCGAGGAGAGCAAGACCCTGATCGTGCTGTTCATCGATGAGATCCACATGCTGATGGGCAACGGCAAGGATGATGCCGCCAACATCCTGAAGCCAGCCCTGAGCCGCGGACAGCTGAAGGTCATCGGAGCCACCACCAACAACGAGTACCGCAGCATCGTGGAGAAGGATGGAGCCTTCGAGCGCCGCTTCCAGAAGATCGAGGTGGCCGAGCCAAGCGTGCGCCAGACCGTGGCCATCCTGCGCGGACTGCAGCCCAAGTACGAGATCCACCACGGCGTGCGCATCCTGGATAGCGCCCTGGTGACCGCCGCCCAGCTGGCCAAGCGCTACCTGCCATACCGCCGCCTGCCAGATAGCGCCCTGGATCTGGTGGATATCAGCTGCGCCGGAGTGGCCGTGGCCCGCGATAGCAAGCCAGAGGAGCTGGATAGCAAGGAGCGCCAGCTGCAGCTGATCCAGGTGGAGATCAAGGCCCTGGAGCGCGATGAGGATGCCGATAGCACCACCAAGGATCGCCTGAAGCTGGCCCGCCAGAAGGAGGCCAGCCTGCAGGAGGAGCTGGAGCCACTGCGCCAGCGCTACAACGAGGAGAAGCACGGCCACGAGGAGCTGACCCAGGCTAAGAAAAAGCTGGATGAGCTGGAGAACAAGGCCCTGGATGCCGAGCGCCGCTACGATACCGCCACCGCCGCCGATCTGCGCTACTTCGCCATCCCCGATATCAAGAAGCAGATCGAGAAGCTGGAGGATCAGGTGGCCGAGGAGGAGCGCCGCGCCGGCGCCAACAGCATGATCCAGAACGTGGTGGATAGCGATACCATCAGCGAGACCGCCGCCCGCCTGACCGGCATCCCCGTGAAGAAGCTGAGCGAGAGCGAGAACGAGAAGCTGATCCACATGGAGCGCGATCTGAGCAGCGAGGTGGTGGGCCAGATGGATGCCATCAAGGCCGTGAGCAACGCCGTGCGCCTGAGCCGCAGCGGACTGGCCAACCCACGCCAGCCAGCCAGCTTCCTGTTCCTGGGCCTGAGCGGCAGCGGCAAGACCGAGCTGGCCAAGAAGGTGGCCGGCTTCCTGTTCAACGATGAGGATATGATGATCCGCGTGGATTGCAGCGAGCTGAGCGAGAAGTACGCCGTGAGCAAGCTGCTGGGCACCACCGCCGGCTACGTGGGCTACGATGAGGGCGGCTTCCTGACCAACCAGCTGCAGTACAAGCCCTACAGCGTGCTGCTGTTCGATGAGGTGGAGAAGGCCCACCCCGATGTGCTGACCGTGATGCTGCAGATGCTGGATGATGGCCGCATCACCAGCGGCCAGGGCAAGACCATCGATTGCAGCAACTGCATCGTGATCATGACCAGCAACCTGGGCGCCGAGTTCATCAACAGCCAGCAGGGCAGCAAGATCCAGGAGAGCACCAAGAACCTGGTCATGGGCGCCGTGCGCCAGCACTTCCGCCCCGAGTTCCTGAACCGCATCAGCAGCATCGTGATCTTCAACAAGCTGAGCCGCAAGGCCATCCACAAGATCGTGGATATCCGCCTGAAGGAGATTGAGGAGCGCTTCGAGCAGAACGATAAGCACTACAAGCTGAACCTGACCCAGGAGGCCAAGGATTTCCTGGCCAAGTACGGCTACAGCGATGATATGGGCGCCCGCCCCCTGAACCGCCTGATCCAGAACGAGATCCTGAACAAGCTGGCCCTGCGCATCCTGAAGAACGAGATCAAGGATAAGGAGACCGTGAACGTGGTGCTGAAGAAGGGCAAGAGCCGCGATGAGAACGTGCCAGAGGAGGCCGAGGAGTGCCTGGAGGTGCTGCCAAACCACGAGGCCACCATCGGAGCCGATACCCTGGGCGATGATGATAACGAGGATAGCATGGAGATCGATGATGATCTGGATTAA5′-TCGAACCCAGTGGAAACCCTTGAAATGCCTTTAACTCGAGACGG-3′ and 5′-GTACCCGTCTCGAGTTAAAGGCATTTCAAGGGTTTCCACTGGGT-3′), with a single copy of the 31 bp glass-binding site from the Rh1 proximal enhancer, were duplexed and ligated into the vector, producing p1×GR (1 copy of glass reporter). This plasmid was then modified to introduce the GAL4 coding sequence, excised from pGaTN Multiple insertion lines were characterized for each transgene. To create the 1×gr-GAL4 driver line, the pGMR (glass multimer reporter) vector 2)  area was selected; particle analysis was performed with ImageJ and statistics performed with one-way ANOVA and unpaired t-test.Heads were frozen in Tissue Freezing Medium (Electron Microscopy Sciences) and sectioned at 12 µm by cryotome, and the tissue sections were then fixed with 4% paraformaldehyde. Immunohistochemistry was performed according to standard procedures using primary antibodies anti-HA 5B1D10  or anti-myc 9E10  (both mouse) alongside either anti-Hsp104  or anti-Hsp70  (both rabbit). Hsp70 staining was confirmed with human-specific anti-Hsp70  (mouse) alongside anti-HA Y11  or anti-myc A14  (both rabbit). Rabbit primary antibodies were preadsorbed at 1∶25 with fixed, dissected wild-type larvae. Secondary antibodies were Alexa Fluor 594 Goat-anti-Mouse IgG , Alexa Fluor 488 Goat-anti-Rabbit IgG , Alexa Fluor 594 Goat-anti-Rabbit IgG , and Alexa Fluor 488 Goat-anti-Mouse IgG . Sections were co-stained with Hoechst nuclear dye  and viewed with a Leica fluorescence microscope. A 75 µm×75 µm square  and anti-actin  with secondary antibody Goat-anti-Rabbit-HRP . For MJD aggregation analysis through SDD-AGE (Semi-Denaturing Detergent Agarose Gel Electrophoresis) and accompanying Western immunoblots, heads were ground in lysis buffer  A 4.0 mg/ml stock solution of RU486 (Sigma M8046) was prepared in 100% ethanol, and then 50 µl (200 µg) was added to pre-prepared food vials containing ∼12 ml of food and gently shaken overnight