Caracterización del transcriptoma de la araña polimórfica de color Gasteracantha cacriformis, con enfoque a los genes de pigmento

El polimorfismo de coloración es un sistema útil para estudiar procesos evolutivos y de desarrollo. No obstante, el estudio de este rasgo se ha sesgado a organismos modelo y la coloración en arácnidos no ha sido bien estudiada. La araña Gasteracantha cancriformis es una especie neotropical que prese...

Full description

Autores:
Tipo de recurso:
Fecha de publicación:
2020
Institución:
Universidad del Rosario
Repositorio:
Repositorio EdocUR - U. Rosario
Idioma:
spa
OAI Identifier:
oai:repository.urosario.edu.co:10336/28197
Acceso en línea:
https://doi.org/10.48713/10336_28197
https://repository.urosario.edu.co/handle/10336/28197
Palabra clave:
Gasteracantha cancriformis
Transcriptoma
Expresión diferencial de genes
Polimorfismo de color
Invertebrados
Evolución & genética
Gasteracantha cancriformis
transcriptome
differential gene expression
color polymorphism
Gasteracantha cancriformis
Transcriptome
Differential gene expression
Color polymorphism
Rights
License
Atribución-NoComercial-SinDerivadas 2.5 Colombia
id EDOCUR2_a1e759c5449b2b5039716a800c595757
oai_identifier_str oai:repository.urosario.edu.co:10336/28197
network_acronym_str EDOCUR2
network_name_str Repositorio EdocUR - U. Rosario
repository_id_str
dc.title.spa.fl_str_mv Caracterización del transcriptoma de la araña polimórfica de color Gasteracantha cacriformis, con enfoque a los genes de pigmento
dc.title.TranslatedTitle.eng.fl_str_mv Characterization of the transcriptome of color-polymorphic spider Gasteracantha cancriformis with special reference to pigment genes.
title Caracterización del transcriptoma de la araña polimórfica de color Gasteracantha cacriformis, con enfoque a los genes de pigmento
spellingShingle Caracterización del transcriptoma de la araña polimórfica de color Gasteracantha cacriformis, con enfoque a los genes de pigmento
Gasteracantha cancriformis
Transcriptoma
Expresión diferencial de genes
Polimorfismo de color
Invertebrados
Evolución & genética
Gasteracantha cancriformis
transcriptome
differential gene expression
color polymorphism
Gasteracantha cancriformis
Transcriptome
Differential gene expression
Color polymorphism
title_short Caracterización del transcriptoma de la araña polimórfica de color Gasteracantha cacriformis, con enfoque a los genes de pigmento
title_full Caracterización del transcriptoma de la araña polimórfica de color Gasteracantha cacriformis, con enfoque a los genes de pigmento
title_fullStr Caracterización del transcriptoma de la araña polimórfica de color Gasteracantha cacriformis, con enfoque a los genes de pigmento
title_full_unstemmed Caracterización del transcriptoma de la araña polimórfica de color Gasteracantha cacriformis, con enfoque a los genes de pigmento
title_sort Caracterización del transcriptoma de la araña polimórfica de color Gasteracantha cacriformis, con enfoque a los genes de pigmento
dc.contributor.advisor.none.fl_str_mv Pardo Díaz, Geimy Carolina
dc.subject.spa.fl_str_mv Gasteracantha cancriformis
Transcriptoma
Expresión diferencial de genes
Polimorfismo de color
topic Gasteracantha cancriformis
Transcriptoma
Expresión diferencial de genes
Polimorfismo de color
Invertebrados
Evolución & genética
Gasteracantha cancriformis
transcriptome
differential gene expression
color polymorphism
Gasteracantha cancriformis
Transcriptome
Differential gene expression
Color polymorphism
dc.subject.ddc.spa.fl_str_mv Invertebrados
Evolución & genética
dc.subject.keyword.eng.fl_str_mv Gasteracantha cancriformis
transcriptome
differential gene expression
color polymorphism
dc.subject.keyword.spa.fl_str_mv Gasteracantha cancriformis
Transcriptome
Differential gene expression
Color polymorphism
description El polimorfismo de coloración es un sistema útil para estudiar procesos evolutivos y de desarrollo. No obstante, el estudio de este rasgo se ha sesgado a organismos modelo y la coloración en arácnidos no ha sido bien estudiada. La araña Gasteracantha cancriformis es una especie neotropical que presenta polimorfismo de coloración abdominal con al menos 16 morfos conocidos distribuidos desde el sur de Estados Unidos hasta el norte de Argentina. A la fecha, las bases genéticas que controlan dicho polimorfismo son desconocidas. En este estudio implementé RNAseq para ensamblar el transcriptoma de Gasteracantha, y evaluar expresión génica diferencial asociada a la coloración en hembras de color blanco, amarillo y naranja. A partir de las secuencias de los transcritos hice análisis nucleotídicos y estadísticos para establecer si existe una relación entre coloración y polimorfismos de ADN (SNPs) en el transcriptoma. En general, el perfil de expresión génica fue similar entre los fenotipos amarillo y naranja, quienes difirieron del fenotipo blanco. En los morfos amarillo y naranja dectecté mayor expresión de genes relacionados a astacina y vitelogenina, lo cual sugiere que estas coloraciones en G. cancriformis son producto de carotenoides. A nivel nucleotídico encontré que los transcritos diferencialmente expresados muestran señal asociada a fenotipo de color, pero esto no sucede a nivel de transcriptoma completo. Los datos obtenidos en este estudio proporcionan un recurso genético valioso para futuras investigaciones en arañas neotropicales y constituyen un paso fundamental hacia la identificación de las bases genéticas del polimorfismo de color en estos animales.
publishDate 2020
dc.date.accessioned.none.fl_str_mv 2020-08-20T17:52:20Z
dc.date.available.none.fl_str_mv 2020-08-20T17:52:20Z
dc.date.created.none.fl_str_mv 2020-07-24
dc.type.eng.fl_str_mv bachelorThesis
dc.type.coar.fl_str_mv http://purl.org/coar/resource_type/c_7a1f
dc.type.document.spa.fl_str_mv Monografía
dc.type.spa.spa.fl_str_mv Trabajo de grado
dc.identifier.doi.none.fl_str_mv https://doi.org/10.48713/10336_28197
dc.identifier.uri.none.fl_str_mv https://repository.urosario.edu.co/handle/10336/28197
url https://doi.org/10.48713/10336_28197
https://repository.urosario.edu.co/handle/10336/28197
dc.language.iso.spa.fl_str_mv spa
language spa
dc.rights.spa.fl_str_mv Atribución-NoComercial-SinDerivadas 2.5 Colombia
Atribución-NoComercial-SinDerivadas 2.5 Colombia
dc.rights.coar.fl_str_mv http://purl.org/coar/access_right/c_abf2
dc.rights.acceso.spa.fl_str_mv Abierto (Texto Completo)
dc.rights.uri.none.fl_str_mv http://creativecommons.org/licenses/by-nc-nd/2.5/co/
rights_invalid_str_mv Atribución-NoComercial-SinDerivadas 2.5 Colombia
Abierto (Texto Completo)
http://creativecommons.org/licenses/by-nc-nd/2.5/co/
http://purl.org/coar/access_right/c_abf2
dc.format.mimetype.none.fl_str_mv application/pdf
dc.publisher.spa.fl_str_mv Universidad del Rosario
dc.publisher.department.spa.fl_str_mv Facultad de Ciencias Naturales y Matemáticas
dc.publisher.program.spa.fl_str_mv Biología
institution Universidad del Rosario
dc.source.bibliographicCitation.spa.fl_str_mv Ando, S., Takeyama, T., & Hatano, M. (1986). Transport associated with serum vitellogenin of carotenoid in chum salmon (Oncorhynchus ketaf. Agricultural and Biological Chemistry, 50(3), 557–563. https://doi.org/10.1080/00021369.1986.10867435
Andrews, S. (2010). FastQC A Quality Control tool for High Throughput Sequence Data. Babraham Bioinformatics. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
Bazyar Lakeh, A. A., Ahmadi, M. R., Safi, S., Ytrestøyl, T., & Bjerkeng, B. (2010). Growth performance, mortality and carotenoid pigmentation of fry offspring as affected by dietary supplementation of astaxanthin to female rainbow trout (Oncorhynchus mykiss) broodstock. Journal of Applied Ichthyology, 26(1), 35–39. https://doi.org/10.1111/j.1439-0426.2009.01349.x
Bolger, A., Lohse, M., & Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30(15), 2114–2120. https://doi.org/10.1093/bioinformatics/btu170
Bond, J. S., & Beynon, R. J. (1995). The astacin family of metalloendopeptidases. In Protein Science (Vol. 4, Issue 7, pp. 1247–1261). John Wiley & Sons, Ltd. https://doi.org/10.1002/pro.5560040701
Bukowski, T. C., Linn, C. D., & Christenson, T. E. (2001). Copulation and sperm release in Gasteracantha cancriformis (Araneae: Araneidae): Differential male behaviour based on female mating history. Animal Behaviour, 62(5), 887–895. https://doi.org/10.1006/anbe.2001.1834
Chamberland, L., Salgado-Roa, F. C., Basco, A., Crastz-Flores, A., Binford, G. J., & Agnarsson, I. (2020). Phylogeography of the widespread Caribbean spiny orb weaver Gasteracantha cancriformis. PeerJ, 8, e8976. https://doi.org/10.7717/peerj.8976
Coddington, J. A., Griswold, C. E., Davila, D. S., Peftaranda, E., & Larcher, S. F. (1991). Designing and Testing Sampling Protocols to Estimate Biodiversity in Tropical Ecosystems. Dioscorides Press.
Conesa, A., Götz, S., García-Gómez, J. M., Terol, J., Talón, M., & Robles, M. (2005). Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bio, 21(18), 3674–3676. https://doi.org/10.1093/bioinformatics/bti610
Croucher, P. J. P., Brewer, M. S., Winchell, C. J., Oxford, G. S., & Gillespie, R. G. (2013). De novo characterization of the gene-rich transcriptomes of two color-polymorphic spiders, Theridion grallator and T. californicum (Araneae: Theridiidae), with special reference to pigment genes. BMC Genomics, 14(1), 1–18. https://doi.org/10.1186/1471-2164-14-862
Danecek, P., Auton, A., Abecasis, G., Albers, C. A., Banks, E., Depristo, M. A., Handsaker, R. E., Lunter, G., Marth, G. T., Sherry, S. T., Mcvean, G., Durbin, R., & Project, G. (2011). The variant call format and VCFtools. Bioinformatics, 27(15), 2156–2158. https://doi.org/10.1093/bioinformatics/btr330
Foradori, M. J., Tillinghast, E. K., Smith, J. S., Townley, M. A., & Mooney, R. E. (2006). Astacin family metallopeptidases and serine peptidase inhibitors in spider digestive fluid. Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology, 143(3), 257–268. https://doi.org/10.1016/j.cbpb.2005.08.012
García, M., & Lara, M. (2013). The use of carotenoid in aquaculture. Research Journal of Fisheries and Hydrobiology, 8(2), 38–49.
Gawryszewski, F. M., & Motta, P. C. (2012). Colouration of the orb-web spider Gasteracantha cancriformis does not increase its foraging success. Ethology Ecology and Evolution, 24(1), 23–38. https://doi.org/10.1080/03949370.2011.582044
Gawryszewski, Felipe M., & Motta, P. C. (2008). The silk tuft web decorations of the orb-weaver Gasteracantha cancriformis: Testing the prey attraction and the web advertisement hypotheses. Behaviour, 145(3), 277–295. https://doi.org/10.1163/156853908783402911
Gray, S. M., & McKinnon, J. S. (2007). Linking color polymorphism maintenance and speciation. In Trends in Ecology and Evolution (Vol. 22, Issue 2, pp. 71–79). https://doi.org/10.1016/j.tree.2006.10.005
Gross, J. B., & Wilkens, H. (2013). Albinism in phylogenetically and geographically distinct populations of Astyanax cavefish arises through the same loss-of-function Oca2 allele. Heredity, 111(2), 122–130. https://doi.org/10.1038/hdy.2013.26
Haas, B. J., Papanicolaou, A., Yassour, M., Grabherr, M., Blood, P. D., Bowden, J., Couger, M. B., Eccles, D., Li, B., Lieber, M., Macmanes, M. D., Ott, M., Orvis, J., Pochet, N., Strozzi, F., Weeks, N., Westerman, R., William, T., Dewey, C. N., … Regev, A. (2013). De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nature Protocols, 8(8), 1494–1512. https://doi.org/10.1038/nprot.2013.084
Hoekstra, H. E., Hirschmann, R. J., Bundey, R. A., Insel, P. A., & Crossland, J. P. (2006). A single amino acid mutation contributes to adaptive beach mouse color pattern. Science, 313(5783), 101–104. https://doi.org/10.1126/science.1126121
Hsiung, B. K., Justyn, N. M., Blackledge, T. A., & Shawkey, M. D. (2017). Spiders have rich pigmentary and structural colour palettes. Journal of Experimental Biology, 220(11), 1975–1983. https://doi.org/10.1242/jeb.156083
Khalaila, I., Peter-Katalinic, J., Tsang, C., Radcliffe, C. M., Aflalo, E. D., Harvey, D. J., Dwek, R. A., Rudd, P. M., & Sagi, A. (2004). Structural characterization of the N-glycan moiety and site of glycosylation in vitellogenin from the decapod crustacean Cherax quadricarinatus. Glycobiology, 14(9), 767–774. https://doi.org/10.1093/glycob/cwh105
Krishnan, M., Bharathiraja, C., Warrier, S., Krishnan, M., Muthumeenakshi, P., Bharathiraja, C., & Subramoniam, T. (2008). A comparative study on vitellogenin receptor of a lepidopteran insect (Spodoptera litura) and a decapod crustacean (Scylla serrata): Phylogenetic implication and co-evolution with vitellogenins. In J Endocrinol Reprod (Vol. 12). https://www.researchgate.net/publication/241727062
Kronforst, M. R., Barsh, G. S., Kopp, A., Mallet, J., Monteiro, A., Mullen, S. P., Protas, M., Rosenblum, E. B., Schneider, C. J., & Hoekstra, H. E. (2012). Unraveling the thread of nature’s tapestry: The genetics of diversity and convergence in animal pigmentation. Pigment Cell and Melanoma Research, 25(4), 411–433. https://doi.org/10.1111/j.1755-148X.2012.01014.x
Kronforst, M. R., & Papa, R. (2015). The functional basis of wing patterning in Heliconius butterflies: The molecules behind mimicry. In Genetics (Vol. 200, Issue 1, pp. 1–19). Genetics. https://doi.org/10.1534/genetics.114.172387
Langmead, B., Trapnell, C., Pop, M., & Salzberg, S. L. (2009). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biology, 10(3), R25. https://doi.org/10.1186/gb-2009-10-3-r25
Lefort, V., Desper, R., Gascuel, O., & Rosenberg, M. (2015). FastME 2.0: A Comprehensive, Accurate, and Fast Distance-Based Phylogeny Inference Program. Molecular Biology and Evolution, 32(10), 2798–2800. https://doi.org/10.1093/molbev/msv150
Levi, H. (1978). The American orb-weaver genera colphepeira, micrathena and gasteracantha north of Mexico (Araneae, Araneidae). Bulletin of the Museum of Comparative Zoology, 148(9), 417–442. https://doi.org/10.1038/193728a0
Li, B., & Dewey, C. N. (2011). RSEM: Accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics, 12(1), 323. https://doi.org/10.1186/1471-2105-12-323
Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R., Project, G., & Subgroup, D. P. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics Applications Note, 25(16), 2078–2079. https://doi.org/10.1093/bioinformatics/btp352
Li, Z., Zhang, S., & Liu, Q. (2008). Vitellogenin Functions as a Multivalent Pattern Recognition Receptor with an Opsonic Activity. PLoS ONE, 3(4), e1940. https://doi.org/10.1371/journal.pone.0001940
Liang, Y., Bai, D., Yang, G., Wei, D., Guo, M., Yan, S., Wu, X., & Ning, B. (2012). Effect of Astacin on Growth and Color Formation of Juvenile Red-White Ornamental Carp (Cyprinus carpio var. koi L). The Israeli Journal of Aquaculture, 64. http://www.aquaculturehub.org
Library Prep and Array Kit Selector. (n.d.). Retrieved August 3, 2020, from https://www.illumina.com/library-prep-array-kit-selector.html
Linnen, C. R., Poh, Y. P., Peterson, B. K., Barrett, R. D. H., Larson, J. G., Jensen, J. D., & Hoekstra, H. E. (2013). Adaptive evolution of multiple traits through multiple mutations at a single gene. Science, 339(6125), 1312–1316. https://doi.org/10.1126/science.1233213
Lubzens, E., Lissauer, L., Levavi-Sivan, B., Avarre, J. C., & Sammar, M. (2003). Carotenoid and retinoid transport to fish oocytes and eggs: What is the role of retinol binding protein? In Molecular Aspects of Medicine (Vol. 24, Issue 6, pp. 441–457). Elsevier Ltd. https://doi.org/10.1016/S0098-2997(03)00040-2
Medina, R., Guerra, C., de Almeida, S., Costal, F., Alves, P., Carmo, O., Ferreyra, A., Bonilla, C., Gonzalez, E. E., Kalapothakis, E., & Chávez, C. (2019). Diversity of astacin-like metalloproteases identified by transcriptomic analysis in Peruvian Loxosceles laeta spider venom and in vitro activity characterization. Biochimie, 167, 81–92. https://doi.org/10.1016/j.biochi.2019.08.017
Muma, M. H. (1971). Biological and Behavioral Notes on Gasteracantha cancriformis (Arachnida: Araneidae). In The Florida Entomologist (Vol. 54, Issue 4, p. 345). https://doi.org/10.2307/3493600
Muma, M. H., & Stone, K. J. (1971). Predation of Gasteracantha cancriformis (Arachnidae: Araneidae) Eggs in Florida Citrus Groves by Phalacrotophora epeirae (Insecta: Phoridae) and Arachnophaga ferruginea (Insecta: Eupelmidae). In The Florida Entomologist (Vol. 54, Issue 4, p. 305). https://doi.org/10.2307/3493590
Nachman, M. W., Hoekstra, H. E., & D’Agostino, S. L. (2003). The genetic basis of adaptive melanism in pocket mice. Proceedings of the National Academy of Sciences of the United States of America, 100(9), 5268–5273. https://doi.org/10.1073/pnas.0431157100
Orteu, A., & Jiggins, C. D. (2020). The genomics of coloration provides insights into adaptive evolution. Nature Reviews Genetics. https://doi.org/10.1038/s41576-020-0234-z
Oxford, G. S., & Gillespie, R. G. (1998). Evolution and Ecology of Spider Coloration. Annual Review of Entomology, 43(1), 619–643. https://doi.org/10.1146/annurev.ento.43.1.619
Prosdocimi, F., Bittencourt, D., da Silva, F. R., Kirst, M., Motta, P. C., & Rech, E. L. (2011). Spinning Gland Transcriptomics from Two Main Clades of Spiders (Order: Araneae) - Insights on Their Molecular, Anatomical and Behavioral Evolution. PLoS ONE, 6(6), e21634. https://doi.org/10.1371/journal.pone.0021634
Purcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M. A. R., Bender, D., Maller, J., Sklar, P., De Bakker, P. I. W., Daly, M. J., & Sham, P. C. (2007). PLINK: A Tool Set for Whole-Genome Association and Population-Based Linkage Analyses. Am. J. Hum. Genet, 81, 559–575. https://doi.org/10.1086/519795
Reed, R. D., Papa, R., Martin, A., Hines, H. M., Counterman, B. A., Pardo-Diaz, C., Jiggins, C. D., Chamberlain, N. L., Kronforst, M. R., Chen, R., Halder, G., Nijhout, H. F., & McMillan, W. O. (2011). Optix drives the repeated convergent evolution of butterfly wing pattern mimicry. Science, 333(6046), 1137–1141. https://doi.org/10.1126/science.1208227
Robinson, M. D., Mccarthy, D. J., & Smyth, G. K. (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics Applications Note, 26(1), 139–140. https://doi.org/10.1093/bioinformatics/btp616
Lourenĉo, A. P., Martins, J. R., Bitondi, M. M. G., & Simões, Z. L. P. (2009). Trade-off between immune stimulation and expression of storage protein genes. Archives of Insect Biochemistry and Physiology, 71(2), 70–87. https://doi.org/10.1002/arch.20301
Salgado-Roa, F. C., Pardo-Diaz, C., Lasso, E., Arias, C. F., Solferini, V. N., & Salazar, C. (2018). Gene flow and Andean uplift shape the diversification of Gasteracantha cancriformis (Araneae: Araneidae) in Northern South America. Ecology and Evolution, 8(14), 7131–7142. https://doi.org/10.1002/ece3.4237
Streelman, J. T., Peichel, C. L., & Parichy, D. M. (2007). Developmental Genetics of Adaptation in Fishes: The Case for Novelty. Annual Review of Ecology, Evolution, and Systematics, 38(1), 655–681. https://doi.org/10.1146/annurev.ecolsys.38.091206.095537
Vieira, F. G., Lassalle, F., Korneliussen, T. S., & Fumagalli, M. (2016). Improving the estimation of genetic distances from Next-Generation Sequencing data. Biological Journal of the Linnean Society, 117(1), 139–149. https://doi.org/10.1111/bij.12511
Walter, A., Bechsgaard, J., Scavenius, C., Dyrlund, T. S., Sanggaard, K. W., Enghild, J. J., & Bilde, T. (2017). Characterisation of protein families in spider digestive fluids and their role in extra-oral digestion. BMC Genomics, 18(1), 600. https://doi.org/10.1186/s12864-017-3987-9
White, T. E., & Kemp, D. J. (2015). Technicolour deceit: A sensory basis for the study of colour-based lures. Animal Behaviour, 105, 231–243. https://doi.org/10.1016/j.anbehav.2015.04.025
Wittkopp, P. J., Smith-Winberry, G., Arnold, L. L., Thompson, E. M., Cooley, A. M., Yuan, D. C., Song, Q., & McAllister, B. F. (2011). Local adaptation for body color in Drosophila americana. Heredity, 106(4), 592–602. https://doi.org/10.1038/hdy.2010.90
Wittkopp, Patricia J., & Beldade, P. (2009). Development and evolution of insect pigmentation: Genetic mechanisms and the potential consequences of pleiotropy. Seminars in Cell and Developmental Biology, 20(1), 65–71. https://doi.org/10.1016/j.semcdb.2008.10.002
Xing, L., Sun, L., Liu, S., Wan, Z., Li, X., Miao, T., Zhang, L., Bai, Y., & Yang, H. (2018). Growth, histology, ultrastructure and expression of MITF and astacin in the pigmentation stages of green, white and purple morphs of the sea cucumber, Apostichopus japonicus. Aquaculture Research, 49(1), 177–187. https://doi.org/10.1111/are.13446
Yoshida, M. (1989). Predatory behavior of gasteracantha mammosa C. koch (araneae: Araneidae). Acta Arachnologica, 37(2), 57–67. https://doi.org/10.2476/asjaa.37.57
dc.source.instname.spa.fl_str_mv instname:Universidad del Rosario
dc.source.reponame.spa.fl_str_mv reponame:Repositorio Institucional EdocUR
bitstream.url.fl_str_mv https://repository.urosario.edu.co/bitstreams/bc977a25-d48c-40b5-8196-f54ee290b76f/download
https://repository.urosario.edu.co/bitstreams/97f9b9ae-789f-44d1-9493-4e86be9f3884/download
https://repository.urosario.edu.co/bitstreams/f61cb0b8-8c7b-482d-932d-38eeeb6ca70c/download
https://repository.urosario.edu.co/bitstreams/11281199-1995-42ca-90d2-53c660140a27/download
https://repository.urosario.edu.co/bitstreams/f25cd923-3d6b-4d89-82f4-5bf1fac96175/download
bitstream.checksum.fl_str_mv 1cc924e28f18f5060febfe1601abf7b2
fab9d9ed61d64f6ac005dee3306ae77e
217700a34da79ed616c2feb68d4c5e06
b79f69868177fabf1c584117d4a97752
fa58a35112b6756249ae64b1299a7256
bitstream.checksumAlgorithm.fl_str_mv MD5
MD5
MD5
MD5
MD5
repository.name.fl_str_mv Repositorio institucional EdocUR
repository.mail.fl_str_mv edocur@urosario.edu.co
_version_ 1814167633899028480
spelling Pardo Díaz, Geimy Carolina53107311600Torres Quintero, Paula AlexandraBiólogoFull timeed70e844-feeb-4051-b578-f95d99cc7e236002020-08-20T17:52:20Z2020-08-20T17:52:20Z2020-07-24El polimorfismo de coloración es un sistema útil para estudiar procesos evolutivos y de desarrollo. No obstante, el estudio de este rasgo se ha sesgado a organismos modelo y la coloración en arácnidos no ha sido bien estudiada. La araña Gasteracantha cancriformis es una especie neotropical que presenta polimorfismo de coloración abdominal con al menos 16 morfos conocidos distribuidos desde el sur de Estados Unidos hasta el norte de Argentina. A la fecha, las bases genéticas que controlan dicho polimorfismo son desconocidas. En este estudio implementé RNAseq para ensamblar el transcriptoma de Gasteracantha, y evaluar expresión génica diferencial asociada a la coloración en hembras de color blanco, amarillo y naranja. A partir de las secuencias de los transcritos hice análisis nucleotídicos y estadísticos para establecer si existe una relación entre coloración y polimorfismos de ADN (SNPs) en el transcriptoma. En general, el perfil de expresión génica fue similar entre los fenotipos amarillo y naranja, quienes difirieron del fenotipo blanco. En los morfos amarillo y naranja dectecté mayor expresión de genes relacionados a astacina y vitelogenina, lo cual sugiere que estas coloraciones en G. cancriformis son producto de carotenoides. A nivel nucleotídico encontré que los transcritos diferencialmente expresados muestran señal asociada a fenotipo de color, pero esto no sucede a nivel de transcriptoma completo. Los datos obtenidos en este estudio proporcionan un recurso genético valioso para futuras investigaciones en arañas neotropicales y constituyen un paso fundamental hacia la identificación de las bases genéticas del polimorfismo de color en estos animales.Color polymorphism in animals is a useful system to study evolutionary and developmental processes. However, the study of this trait is biased towards model organisms, while coloration in arachnids is less explored. Gasteracantha cancriformis is a neotropical spider that displays color polymorphism with at least 16 known morphs distributed from the southern United States to northern Argentina but, to date, the genetic basis underlying such polymorphism is unknown. In this study, I used RNAseq to generate a transcriptome assembly for Gasteracantha and assess differential gene expression between three different color morphs: white, yellow and orange. I also explored whether single nucleotide polymorphism (SNPs) across the transcriptome display any association with coloration. Overall, the gene expression profile of the yellow and orange morphs was similar, and they both differed from the white morph. Consistently, at nucleotide level I found that SNPs in the differential expressed transcripts cluster orange and yellow morphs together while the white morph appears more differentiated. Also, I detected higher expression levels of astacin and vitellogenin genes in the yellow and orange morphs, suggesting that these colorations in G. cancriformis are due to carotenoids. The data obtained in this study provide a valuable genetic resource for future studies on neotropical spiders and constitute a step towards the identification of the genetic basis of color polymorphism in these animals.application/pdfhttps://doi.org/10.48713/10336_28197 https://repository.urosario.edu.co/handle/10336/28197spaUniversidad del RosarioFacultad de Ciencias Naturales y MatemáticasBiologíaAtribución-NoComercial-SinDerivadas 2.5 ColombiaAtribución-NoComercial-SinDerivadas 2.5 ColombiaAbierto (Texto Completo)EL AUTOR, manifiesta que la obra objeto de la presente autorización es original y la realizó sin violar o usurpar derechos de autor de terceros, por lo tanto la obra es de exclusiva autoría y tiene la titularidad sobre la misma.http://creativecommons.org/licenses/by-nc-nd/2.5/co/http://purl.org/coar/access_right/c_abf2Ando, S., Takeyama, T., & Hatano, M. (1986). Transport associated with serum vitellogenin of carotenoid in chum salmon (Oncorhynchus ketaf. Agricultural and Biological Chemistry, 50(3), 557–563. https://doi.org/10.1080/00021369.1986.10867435Andrews, S. (2010). FastQC A Quality Control tool for High Throughput Sequence Data. Babraham Bioinformatics. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/Bazyar Lakeh, A. A., Ahmadi, M. R., Safi, S., Ytrestøyl, T., & Bjerkeng, B. (2010). Growth performance, mortality and carotenoid pigmentation of fry offspring as affected by dietary supplementation of astaxanthin to female rainbow trout (Oncorhynchus mykiss) broodstock. Journal of Applied Ichthyology, 26(1), 35–39. https://doi.org/10.1111/j.1439-0426.2009.01349.xBolger, A., Lohse, M., & Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30(15), 2114–2120. https://doi.org/10.1093/bioinformatics/btu170Bond, J. S., & Beynon, R. J. (1995). The astacin family of metalloendopeptidases. In Protein Science (Vol. 4, Issue 7, pp. 1247–1261). John Wiley & Sons, Ltd. https://doi.org/10.1002/pro.5560040701Bukowski, T. C., Linn, C. D., & Christenson, T. E. (2001). Copulation and sperm release in Gasteracantha cancriformis (Araneae: Araneidae): Differential male behaviour based on female mating history. Animal Behaviour, 62(5), 887–895. https://doi.org/10.1006/anbe.2001.1834Chamberland, L., Salgado-Roa, F. C., Basco, A., Crastz-Flores, A., Binford, G. J., & Agnarsson, I. (2020). Phylogeography of the widespread Caribbean spiny orb weaver Gasteracantha cancriformis. PeerJ, 8, e8976. https://doi.org/10.7717/peerj.8976Coddington, J. A., Griswold, C. E., Davila, D. S., Peftaranda, E., & Larcher, S. F. (1991). Designing and Testing Sampling Protocols to Estimate Biodiversity in Tropical Ecosystems. Dioscorides Press.Conesa, A., Götz, S., García-Gómez, J. M., Terol, J., Talón, M., & Robles, M. (2005). Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bio, 21(18), 3674–3676. https://doi.org/10.1093/bioinformatics/bti610Croucher, P. J. P., Brewer, M. S., Winchell, C. J., Oxford, G. S., & Gillespie, R. G. (2013). De novo characterization of the gene-rich transcriptomes of two color-polymorphic spiders, Theridion grallator and T. californicum (Araneae: Theridiidae), with special reference to pigment genes. BMC Genomics, 14(1), 1–18. https://doi.org/10.1186/1471-2164-14-862Danecek, P., Auton, A., Abecasis, G., Albers, C. A., Banks, E., Depristo, M. A., Handsaker, R. E., Lunter, G., Marth, G. T., Sherry, S. T., Mcvean, G., Durbin, R., & Project, G. (2011). The variant call format and VCFtools. Bioinformatics, 27(15), 2156–2158. https://doi.org/10.1093/bioinformatics/btr330Foradori, M. J., Tillinghast, E. K., Smith, J. S., Townley, M. A., & Mooney, R. E. (2006). Astacin family metallopeptidases and serine peptidase inhibitors in spider digestive fluid. Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology, 143(3), 257–268. https://doi.org/10.1016/j.cbpb.2005.08.012García, M., & Lara, M. (2013). The use of carotenoid in aquaculture. Research Journal of Fisheries and Hydrobiology, 8(2), 38–49.Gawryszewski, F. M., & Motta, P. C. (2012). Colouration of the orb-web spider Gasteracantha cancriformis does not increase its foraging success. Ethology Ecology and Evolution, 24(1), 23–38. https://doi.org/10.1080/03949370.2011.582044Gawryszewski, Felipe M., & Motta, P. C. (2008). The silk tuft web decorations of the orb-weaver Gasteracantha cancriformis: Testing the prey attraction and the web advertisement hypotheses. Behaviour, 145(3), 277–295. https://doi.org/10.1163/156853908783402911Gray, S. M., & McKinnon, J. S. (2007). Linking color polymorphism maintenance and speciation. In Trends in Ecology and Evolution (Vol. 22, Issue 2, pp. 71–79). https://doi.org/10.1016/j.tree.2006.10.005Gross, J. B., & Wilkens, H. (2013). Albinism in phylogenetically and geographically distinct populations of Astyanax cavefish arises through the same loss-of-function Oca2 allele. Heredity, 111(2), 122–130. https://doi.org/10.1038/hdy.2013.26Haas, B. J., Papanicolaou, A., Yassour, M., Grabherr, M., Blood, P. D., Bowden, J., Couger, M. B., Eccles, D., Li, B., Lieber, M., Macmanes, M. D., Ott, M., Orvis, J., Pochet, N., Strozzi, F., Weeks, N., Westerman, R., William, T., Dewey, C. N., … Regev, A. (2013). De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nature Protocols, 8(8), 1494–1512. https://doi.org/10.1038/nprot.2013.084Hoekstra, H. E., Hirschmann, R. J., Bundey, R. A., Insel, P. A., & Crossland, J. P. (2006). A single amino acid mutation contributes to adaptive beach mouse color pattern. Science, 313(5783), 101–104. https://doi.org/10.1126/science.1126121Hsiung, B. K., Justyn, N. M., Blackledge, T. A., & Shawkey, M. D. (2017). Spiders have rich pigmentary and structural colour palettes. Journal of Experimental Biology, 220(11), 1975–1983. https://doi.org/10.1242/jeb.156083Khalaila, I., Peter-Katalinic, J., Tsang, C., Radcliffe, C. M., Aflalo, E. D., Harvey, D. J., Dwek, R. A., Rudd, P. M., & Sagi, A. (2004). Structural characterization of the N-glycan moiety and site of glycosylation in vitellogenin from the decapod crustacean Cherax quadricarinatus. Glycobiology, 14(9), 767–774. https://doi.org/10.1093/glycob/cwh105Krishnan, M., Bharathiraja, C., Warrier, S., Krishnan, M., Muthumeenakshi, P., Bharathiraja, C., & Subramoniam, T. (2008). A comparative study on vitellogenin receptor of a lepidopteran insect (Spodoptera litura) and a decapod crustacean (Scylla serrata): Phylogenetic implication and co-evolution with vitellogenins. In J Endocrinol Reprod (Vol. 12). https://www.researchgate.net/publication/241727062Kronforst, M. R., Barsh, G. S., Kopp, A., Mallet, J., Monteiro, A., Mullen, S. P., Protas, M., Rosenblum, E. B., Schneider, C. J., & Hoekstra, H. E. (2012). Unraveling the thread of nature’s tapestry: The genetics of diversity and convergence in animal pigmentation. Pigment Cell and Melanoma Research, 25(4), 411–433. https://doi.org/10.1111/j.1755-148X.2012.01014.xKronforst, M. R., & Papa, R. (2015). The functional basis of wing patterning in Heliconius butterflies: The molecules behind mimicry. In Genetics (Vol. 200, Issue 1, pp. 1–19). Genetics. https://doi.org/10.1534/genetics.114.172387Langmead, B., Trapnell, C., Pop, M., & Salzberg, S. L. (2009). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biology, 10(3), R25. https://doi.org/10.1186/gb-2009-10-3-r25Lefort, V., Desper, R., Gascuel, O., & Rosenberg, M. (2015). FastME 2.0: A Comprehensive, Accurate, and Fast Distance-Based Phylogeny Inference Program. Molecular Biology and Evolution, 32(10), 2798–2800. https://doi.org/10.1093/molbev/msv150Levi, H. (1978). The American orb-weaver genera colphepeira, micrathena and gasteracantha north of Mexico (Araneae, Araneidae). Bulletin of the Museum of Comparative Zoology, 148(9), 417–442. https://doi.org/10.1038/193728a0Li, B., & Dewey, C. N. (2011). RSEM: Accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics, 12(1), 323. https://doi.org/10.1186/1471-2105-12-323Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R., Project, G., & Subgroup, D. P. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics Applications Note, 25(16), 2078–2079. https://doi.org/10.1093/bioinformatics/btp352Li, Z., Zhang, S., & Liu, Q. (2008). Vitellogenin Functions as a Multivalent Pattern Recognition Receptor with an Opsonic Activity. PLoS ONE, 3(4), e1940. https://doi.org/10.1371/journal.pone.0001940Liang, Y., Bai, D., Yang, G., Wei, D., Guo, M., Yan, S., Wu, X., & Ning, B. (2012). Effect of Astacin on Growth and Color Formation of Juvenile Red-White Ornamental Carp (Cyprinus carpio var. koi L). The Israeli Journal of Aquaculture, 64. http://www.aquaculturehub.orgLibrary Prep and Array Kit Selector. (n.d.). Retrieved August 3, 2020, from https://www.illumina.com/library-prep-array-kit-selector.htmlLinnen, C. R., Poh, Y. P., Peterson, B. K., Barrett, R. D. H., Larson, J. G., Jensen, J. D., & Hoekstra, H. E. (2013). Adaptive evolution of multiple traits through multiple mutations at a single gene. Science, 339(6125), 1312–1316. https://doi.org/10.1126/science.1233213Lubzens, E., Lissauer, L., Levavi-Sivan, B., Avarre, J. C., & Sammar, M. (2003). Carotenoid and retinoid transport to fish oocytes and eggs: What is the role of retinol binding protein? In Molecular Aspects of Medicine (Vol. 24, Issue 6, pp. 441–457). Elsevier Ltd. https://doi.org/10.1016/S0098-2997(03)00040-2Medina, R., Guerra, C., de Almeida, S., Costal, F., Alves, P., Carmo, O., Ferreyra, A., Bonilla, C., Gonzalez, E. E., Kalapothakis, E., & Chávez, C. (2019). Diversity of astacin-like metalloproteases identified by transcriptomic analysis in Peruvian Loxosceles laeta spider venom and in vitro activity characterization. Biochimie, 167, 81–92. https://doi.org/10.1016/j.biochi.2019.08.017Muma, M. H. (1971). Biological and Behavioral Notes on Gasteracantha cancriformis (Arachnida: Araneidae). In The Florida Entomologist (Vol. 54, Issue 4, p. 345). https://doi.org/10.2307/3493600Muma, M. H., & Stone, K. J. (1971). Predation of Gasteracantha cancriformis (Arachnidae: Araneidae) Eggs in Florida Citrus Groves by Phalacrotophora epeirae (Insecta: Phoridae) and Arachnophaga ferruginea (Insecta: Eupelmidae). In The Florida Entomologist (Vol. 54, Issue 4, p. 305). https://doi.org/10.2307/3493590Nachman, M. W., Hoekstra, H. E., & D’Agostino, S. L. (2003). The genetic basis of adaptive melanism in pocket mice. Proceedings of the National Academy of Sciences of the United States of America, 100(9), 5268–5273. https://doi.org/10.1073/pnas.0431157100Orteu, A., & Jiggins, C. D. (2020). The genomics of coloration provides insights into adaptive evolution. Nature Reviews Genetics. https://doi.org/10.1038/s41576-020-0234-zOxford, G. S., & Gillespie, R. G. (1998). Evolution and Ecology of Spider Coloration. Annual Review of Entomology, 43(1), 619–643. https://doi.org/10.1146/annurev.ento.43.1.619Prosdocimi, F., Bittencourt, D., da Silva, F. R., Kirst, M., Motta, P. C., & Rech, E. L. (2011). Spinning Gland Transcriptomics from Two Main Clades of Spiders (Order: Araneae) - Insights on Their Molecular, Anatomical and Behavioral Evolution. PLoS ONE, 6(6), e21634. https://doi.org/10.1371/journal.pone.0021634Purcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M. A. R., Bender, D., Maller, J., Sklar, P., De Bakker, P. I. W., Daly, M. J., & Sham, P. C. (2007). PLINK: A Tool Set for Whole-Genome Association and Population-Based Linkage Analyses. Am. J. Hum. Genet, 81, 559–575. https://doi.org/10.1086/519795Reed, R. D., Papa, R., Martin, A., Hines, H. M., Counterman, B. A., Pardo-Diaz, C., Jiggins, C. D., Chamberlain, N. L., Kronforst, M. R., Chen, R., Halder, G., Nijhout, H. F., & McMillan, W. O. (2011). Optix drives the repeated convergent evolution of butterfly wing pattern mimicry. Science, 333(6046), 1137–1141. https://doi.org/10.1126/science.1208227Robinson, M. D., Mccarthy, D. J., & Smyth, G. K. (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics Applications Note, 26(1), 139–140. https://doi.org/10.1093/bioinformatics/btp616Lourenĉo, A. P., Martins, J. R., Bitondi, M. M. G., & Simões, Z. L. P. (2009). Trade-off between immune stimulation and expression of storage protein genes. Archives of Insect Biochemistry and Physiology, 71(2), 70–87. https://doi.org/10.1002/arch.20301Salgado-Roa, F. C., Pardo-Diaz, C., Lasso, E., Arias, C. F., Solferini, V. N., & Salazar, C. (2018). Gene flow and Andean uplift shape the diversification of Gasteracantha cancriformis (Araneae: Araneidae) in Northern South America. Ecology and Evolution, 8(14), 7131–7142. https://doi.org/10.1002/ece3.4237Streelman, J. T., Peichel, C. L., & Parichy, D. M. (2007). Developmental Genetics of Adaptation in Fishes: The Case for Novelty. Annual Review of Ecology, Evolution, and Systematics, 38(1), 655–681. https://doi.org/10.1146/annurev.ecolsys.38.091206.095537Vieira, F. G., Lassalle, F., Korneliussen, T. S., & Fumagalli, M. (2016). Improving the estimation of genetic distances from Next-Generation Sequencing data. Biological Journal of the Linnean Society, 117(1), 139–149. https://doi.org/10.1111/bij.12511Walter, A., Bechsgaard, J., Scavenius, C., Dyrlund, T. S., Sanggaard, K. W., Enghild, J. J., & Bilde, T. (2017). Characterisation of protein families in spider digestive fluids and their role in extra-oral digestion. BMC Genomics, 18(1), 600. https://doi.org/10.1186/s12864-017-3987-9White, T. E., & Kemp, D. J. (2015). Technicolour deceit: A sensory basis for the study of colour-based lures. Animal Behaviour, 105, 231–243. https://doi.org/10.1016/j.anbehav.2015.04.025Wittkopp, P. J., Smith-Winberry, G., Arnold, L. L., Thompson, E. M., Cooley, A. M., Yuan, D. C., Song, Q., & McAllister, B. F. (2011). Local adaptation for body color in Drosophila americana. Heredity, 106(4), 592–602. https://doi.org/10.1038/hdy.2010.90Wittkopp, Patricia J., & Beldade, P. (2009). Development and evolution of insect pigmentation: Genetic mechanisms and the potential consequences of pleiotropy. Seminars in Cell and Developmental Biology, 20(1), 65–71. https://doi.org/10.1016/j.semcdb.2008.10.002Xing, L., Sun, L., Liu, S., Wan, Z., Li, X., Miao, T., Zhang, L., Bai, Y., & Yang, H. (2018). Growth, histology, ultrastructure and expression of MITF and astacin in the pigmentation stages of green, white and purple morphs of the sea cucumber, Apostichopus japonicus. Aquaculture Research, 49(1), 177–187. https://doi.org/10.1111/are.13446Yoshida, M. (1989). Predatory behavior of gasteracantha mammosa C. koch (araneae: Araneidae). Acta Arachnologica, 37(2), 57–67. https://doi.org/10.2476/asjaa.37.57instname:Universidad del Rosarioreponame:Repositorio Institucional EdocURGasteracantha cancriformisTranscriptomaExpresión diferencial de genesPolimorfismo de colorInvertebrados592600Evolución & genética575600Gasteracantha cancriformistranscriptomedifferential gene expressioncolor polymorphismGasteracantha cancriformisTranscriptomeDifferential gene expressionColor polymorphismCaracterización del transcriptoma de la araña polimórfica de color Gasteracantha cacriformis, con enfoque a los genes de pigmentoCharacterization of the transcriptome of color-polymorphic spider Gasteracantha cancriformis with special reference to pigment genes.bachelorThesisMonografíaTrabajo de gradohttp://purl.org/coar/resource_type/c_7a1fORIGINALTorresQuintero-PaulaAlexandra-2020.pdfTorresQuintero-PaulaAlexandra-2020.pdfapplication/pdf1328192https://repository.urosario.edu.co/bitstreams/bc977a25-d48c-40b5-8196-f54ee290b76f/download1cc924e28f18f5060febfe1601abf7b2MD55LICENSElicense.txtlicense.txttext/plain1475https://repository.urosario.edu.co/bitstreams/97f9b9ae-789f-44d1-9493-4e86be9f3884/downloadfab9d9ed61d64f6ac005dee3306ae77eMD56CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://repository.urosario.edu.co/bitstreams/f61cb0b8-8c7b-482d-932d-38eeeb6ca70c/download217700a34da79ed616c2feb68d4c5e06MD57TEXTTorresQuintero-PaulaAlexandra-2020.pdf.txtTorresQuintero-PaulaAlexandra-2020.pdf.txtExtracted texttext/plain54607https://repository.urosario.edu.co/bitstreams/11281199-1995-42ca-90d2-53c660140a27/downloadb79f69868177fabf1c584117d4a97752MD58THUMBNAILTorresQuintero-PaulaAlexandra-2020.pdf.jpgTorresQuintero-PaulaAlexandra-2020.pdf.jpgGenerated Thumbnailimage/jpeg2354https://repository.urosario.edu.co/bitstreams/f25cd923-3d6b-4d89-82f4-5bf1fac96175/downloadfa58a35112b6756249ae64b1299a7256MD5910336/28197oai:repository.urosario.edu.co:10336/281972020-11-21 18:38:38.121http://creativecommons.org/licenses/by-nc-nd/2.5/co/Atribución-NoComercial-SinDerivadas 2.5 Colombiahttps://repository.urosario.edu.coRepositorio institucional EdocURedocur@urosario.edu.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

Publicaciones similares