The midgut transcriptome of Phlebotomus (Larroussius) perniciosus, a vector of Leishmania infantum: comparison of sugar fed and blood fed sand flies

. 2011 May 10 ; 12 () : 223. [epub] 20110510

Jazyk angličtina Země Velká Británie, Anglie Médium electronic

Typ dokumentu srovnávací studie, časopisecké články, Research Support, N.I.H., Intramural, práce podpořená grantem

Perzistentní odkaz   https://www.medvik.cz/link/pmid21569254

Grantová podpora
078937 Wellcome Trust - United Kingdom
Intramural NIH HHS - United States

BACKGROUND: Parasite-vector interactions are fundamental in the transmission of vector-borne diseases such as leishmaniasis. Leishmania development in the vector sand fly is confined to the digestive tract, where sand fly midgut molecules interact with the parasites. In this work we sequenced and analyzed two midgut-specific cDNA libraries from sugar fed and blood fed female Phlebotomus perniciosus and compared the transcript expression profiles. RESULTS: A total of 4111 high quality sequences were obtained from the two libraries and assembled into 370 contigs and 1085 singletons. Molecules with putative roles in blood meal digestion, peritrophic matrix formation, immunity and response to oxidative stress were identified, including proteins that were not previously reported in sand flies. These molecules were evaluated relative to other published sand fly transcripts. Comparative analysis of the two libraries revealed transcripts differentially expressed in response to blood feeding. Molecules up regulated by blood feeding include a putative peritrophin (PperPer1), two chymotrypsin-like proteins (PperChym1 and PperChym2), a putative trypsin (PperTryp3) and four putative microvillar proteins (PperMVP1, 2, 4 and 5). Additionally, several transcripts were more abundant in the sugar fed midgut, such as two putative trypsins (PperTryp1 and PperTryp2), a chymotrypsin (PperChym3) and a microvillar protein (PperMVP3). We performed a detailed temporal expression profile analysis of the putative trypsin transcripts using qPCR and confirmed the expression of blood-induced and blood-repressed trypsins. Trypsin expression was measured in Leishmania infantum-infected and uninfected sand flies, which identified the L. infantum-induced down regulation of PperTryp3 at 24 hours post-blood meal. CONCLUSION: This midgut tissue-specific transcriptome provides insight into the molecules expressed in the midgut of P. perniciosus, an important vector of visceral leishmaniasis in the Old World. Through the comparative analysis of the libraries we identified molecules differentially expressed during blood meal digestion. Additionally, this study provides a detailed comparison to transcripts of other sand flies. Moreover, our analysis of putative trypsins demonstrated that L. infantum infection can reduce the transcript abundance of trypsin PperTryp3 in the midgut of P. perniciosus.

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Killick-Kendrick R. The biology and control of phlebotomine sand flies. Clin Dermatol. 1999;17:279–289. doi: 10.1016/S0738-081X(99)00046-2. PubMed DOI

Kamhawi S. Phlebotomine sand flies and Leishmania parasites: friends or foes? Trends Parasitol. 2006;22:439–445. doi: 10.1016/j.pt.2006.06.012. PubMed DOI

Peacock CS, Seeger K, Harris D, Murphy L, Ruiz JC, Quail MA, Peters N, Adlem E, Tivey A, Aslett M, Kerhornou A, Ivens A, Fraser A, Rajandream MA, Carver T, Norbertczak H, Chillingworth T, Hance Z, Jagels K, Moule S, Ormond D, Rutter S, Squares R, Whitehead S, Rabbinowitsch E, Arrowsmith C, White B, Thurston S, Bringaud F, Baldauf SL, Faulconbridge A, Jeffares D, Depledge DP, Oyola SO, Hilley JD, Brito LO, Tosi LR, Barrell B, Cruz AK, Mottram JC, Smith DF, Berriman M. Comparative genomic analysis of three Leishmania species that cause diverse human disease. Nat Genet. 2007;39:839–847. doi: 10.1038/ng2053. PubMed DOI PMC

Oliveira F, Jochim RC, Valenzuela JG, Kamhawi S. Sand flies, Leishmania, and transcriptome-borne solutions. Parasitol Int. 2009;58:1–5. doi: 10.1016/j.parint.2008.07.004. PubMed DOI PMC

Ramalho-Ortigao M, Jochim RC, Anderson JM, Lawyer PG, Pham VM, Kamhawi S, Valenzuela JG. Exploring the midgut transcriptome of Phlebotomus papatasi: comparative analysis of expression profiles of sugarfed, blood-fed and Leishmania major-infected sandflies. BMC Genomics. 2007. p. 8. PubMed PMC

Jochim RC, Teixeira CR, Laughinghouse A, Mu JB, Oliveira F, Gomes RB, Elnaiem DE, Valenzuela JG. The midgut transcriptome of Lutzomyia longipalpis: comparative analysis of cDNA libraries from sugar-fed, blood-fed, post-digested and Leishmania infantum chagasi-infected sand flies. BMC Genomics. 2008. p. 9. PubMed PMC

Pitaluga AN, Beteille V, Lobo AR, Ortigao-Farias JR, Davila AM, Souza AA, Ramalho-Ortigão JM, Traub-Cseko YM. EST sequencing of blood-fed and Leishmania-infected midgut of Lutzomyia longipalpis, the principal visceral leishmaniasis vector in the Americas. Mol Genet Genomics. 2009;282:307–317. doi: 10.1007/s00438-009-0466-2. PubMed DOI

Ramalho-Ortigao JM, Kamhawi S, Joshi MB, Reynoso D, Lawyer PG, Dwyer DM, Sacks DL, Valenzuela JG. Characterization of a blood activated chitinolytic system in the midgut of the sand fly vectors Lutzomyia longipalpis and Phlebotomus papatasi. Insect Mol Biol. 2005;14:703–712. doi: 10.1111/j.1365-2583.2005.00601.x. PubMed DOI

Kamhawi S, Ramalho-Ortigao M, Pham VM, Kumar S, Lawyer PG, Turco SJ, Barillas-Mury C, Sacks DL, Valenzuela JG. A role for insect galectins in parasite survival. Cell. 2004;119:329–341. doi: 10.1016/j.cell.2004.10.009. PubMed DOI

Sant'Anna MR, Diaz-Albiter H, Mubaraki M, Dillon RJ, Bates PA. Inhibition of trypsin expression in Lutzomyia longipalpis using RNAi enhances the survival of Leishmania. Parasit Vectors. 2009;2:62. doi: 10.1186/1756-3305-2-62. PubMed DOI PMC

Pimenta PFP, Saraiva EMB, Rowton E, Modi GB, Garraway LA, Beverley SM, Turco SJ, Sacks DL. Evidence that the vectorial competence of phlebotomine sand flies for different species of Leishmania is controlled by structural polymorphisms in the surface lipophosphoglycan. Proc Natl Acad Sci USA. 1994;91:9155–9159. doi: 10.1073/pnas.91.19.9155. PubMed DOI PMC

Myskova J, Svobodova M, Beverley SM, Volf P. A lipophosphoglycan-independent development of Leishmania in permissive sand flies. Microbes Infect. 2007;9:317–324. doi: 10.1016/j.micinf.2006.12.010. PubMed DOI PMC

Svarovska A, Ant TH, Seblova V, Jecna L, Beverley SM, Volf P. Leishmania major glycosylation mutants require phosphoglycans (lpg2-) but not lipophosphoglycan (lpg1-) for survival in permissive sand fly vectors. PLoS Negl Trop Dis. 2010;4:e580. doi: 10.1371/journal.pntd.0000580. PubMed DOI PMC

Guo Y, Ribeiro JM, Anderson JM, Bour S. dCAS: a desktop application for cDNA sequence annotation. Bioinformatics. 2009;25:1195–1196. doi: 10.1093/bioinformatics/btp129. PubMed DOI PMC

Ramalho-Ortigao JM, Kamhawi S, Rowton ED, Ribeiro JMC, Valenzuela JG. Cloning and characterization of trypsin- and chymotrypsin-like proteases from the midgut of the sand fly vector Phlebotomus papatasi. Insect Biochem Mol Biol. 2003;33:163–171. doi: 10.1016/S0965-1748(02)00187-X. PubMed DOI

Telleria EL, Pitaluga AN, Ortigao-Farias JR, de Araujo APO, Ramalho-Ortigao JM, Traub-Cseko YM. Constitutive and blood meal-induced trypsin genes in Lutzomyia longipalpis. Arch Insect Biochem Physiol. 2007;66:53–63. doi: 10.1002/arch.20198. PubMed DOI

Dillon RJ, Lane RP. Bloodmeal Digestion in the Midgut of Phlebotomus papatasi and Phlebotomus langeroni. Med Vet Entomol. 1993;7:225–232. doi: 10.1111/j.1365-2915.1993.tb00681.x. PubMed DOI

Schlein Y, Romano H. Leishmania major and L. donovani: effects on proteolytic enzymes of Phlebotomus papatasi (Diptera: Psychodidae) Exp Parasitol. 1986;62:376–380. doi: 10.1016/0014-4894(86)90045-7. PubMed DOI

Dillon RJ, Lane RP. Influence of Leishmania Infection on Blood-Meal Digestion in the Sandflies Phlebotomus-papatasi and P. langeroni. Parasitol Res. 1993;79:492–496. doi: 10.1007/BF00931590. PubMed DOI

Telleria EL, de Araujo AP, Secundino NF, vila-Levy CM, Traub-Cseko YM. Trypsin-Like Serine Proteases in Lutzomyia longipalpis - Expression, Activity and Possible Modulation by Leishmania infantum chagasi. PLoS One. 2010;5:e10697. doi: 10.1371/journal.pone.0010697. PubMed DOI PMC

Graf R, Briegel H. The synthetic pathway of trypsin in the mosquito Aedes aegypti L (Diptera, Culicidae) and in vitro stimulation in isolated midguts. Insect Biochem. 1989;19:129–137. doi: 10.1016/0020-1790(89)90083-8. DOI

Titani K, Ericsson LH, Walsh KA, Neurath H. Amino-acid sequence of bovine carboxypeptidase B. Proc Natl Acad Sci USA. 1975;72:1666–1670. doi: 10.1073/pnas.72.5.1666. PubMed DOI PMC

Lavazec C, Boudin C, Lacroix R, Bonnet S, Diop A, Thiberge S, Boisson B, Tahar R, Bourgoin C. Carboxypeptidases B of Anopheles gambiae as targets for a Plasmodium falciparum transmission-blocking vaccine. Infect Immun. 2007;75:1635–1642. doi: 10.1128/IAI.00864-06. PubMed DOI PMC

Dinglasan RR, Kalume DE, Kanzok SM, Ghosh AK, Muratova O, Pandey A4. et al.Disruption of Plasmodium falciparum development by antibodies against a conserved mosquito midgut antigen. Proc Natl Acad Sci USA. 2007;104:13461–13466. doi: 10.1073/pnas.0702239104. PubMed DOI PMC

Abdullah MA, Valaitis AP, Dean DH. Identification of a Bacillus thuringiensis Cry11Ba toxin-binding aminopeptidase from the mosquito, Anopheles quadrimaculatus. BMC Biochem. 2006;7:16. doi: 10.1186/1471-2091-7-16. PubMed DOI PMC

Pomes A, Melen E, Vailes LD, Retief JD, Arruda LK, Chapman MD. Novel allergen structures with tandem amino acid repeats derived from German and American cockroach. J Biol Chem. 1998;273:30801–30807. doi: 10.1074/jbc.273.46.30801. PubMed DOI

Wittstock U, Agerbirk N, Stauber EJ, Olsen CE, Hippler M, Mitchell-Olds T, Gershenzon J, Vogel H. Successful herbivore attack due to metabolic diversion of a plant chemical defense. Proc Natl Acad Sci USA. 2004;101:4859–4864. doi: 10.1073/pnas.0308007101. PubMed DOI PMC

Fischer HM, Wheat CW, Heckel DG, Vogel H. Evolutionary origins of a novel host plant detoxification gene in butterflies. Mol Biol Evol. 2008;25:809–820. doi: 10.1093/molbev/msn014. PubMed DOI

Abraham EG, Islam S, Srinivasan P, Ghosh AK, Valenzuela JG, Ribeiro JM, Kafatos FC, Dimopoulos G, Jacobs-Lorena M. Analysis of the Plasmodium and Anopheles transcriptional repertoire during ookinete development and midgut invasion. J Biol Chem. 2004;279:5573–5580. PubMed

Shao L, Devenport M, Fujioka H, Ghosh A, Jacobs-Lorena M. Identification and characterization of a novel peritrophic matrix protein, Ae-Aper50, and the microvillar membrane protein, AEG12, from the mosquito, Aedes aegypti. Insect Biochem Molecul Biol. 2005;35:947–959. doi: 10.1016/j.ibmb.2005.03.012. PubMed DOI

Zaidman-Remy A, Herve M, Poidevin M, Pili-Floury S, Kim MS, Blanot D, Oh BD, Ueda R, Mengin-Lecreulx D, Lemaitre B. The Drosophila amidase PGRP-LB modulates the immune response to bacterial infection. Immunity. 2006;24:463–473. doi: 10.1016/j.immuni.2006.02.012. PubMed DOI

Christophides GK, Zdobnov E, Barillas-Mury C, Birney E, Blandin S, Blass C, Brey PT, Collins FH, Danielli A, Dimopoulos G, Hetru C, Hoa NT, Hoffmann JA, Kanzok SM, Letunic I, Levashina EA, Loukeris TG, Lycett G, Meister S, Michel K, Moita LF, Müller HM, Osta MA, Paskewitz SM, Reichhart JM, Rzhetsky A, Troxler L, Vernick KD, Vlachou D, Volz J, von Mering C, Xu J, Zheng L, Bork P, Kafatos FC. Immunity-related genes and gene families in Anopheles gambiae. Science. 2002;298:159–165. doi: 10.1126/science.1077136. PubMed DOI

Meister S, Agianian B, Turlure F, Relogio A, Morlais I, Kafatos FC, Christophides GK. Anopheles gambiae PGRPLC-mediated defense against bacteria modulates infections with malaria parasites. PLoS Pathog. 2009;5:e1000542. doi: 10.1371/journal.ppat.1000542. PubMed DOI PMC

Warr E, Das S, Dong Y, Dimopoulos G. The Gram-negative bacteria-binding protein gene family: its role in the innate immune system of anopheles gambiae and in anti-Plasmodium defence. Insect Mol Biol. 2008;17:39–51. doi: 10.1111/j.1365-2583.2008.00778.x. PubMed DOI

Kumar S, Molina-Cruz A, Gupta L, Rodrigues J, Barillas-Mury C. A peroxidase/dual oxidase system modulates midgut epithelial immunity in Anopheles gambiae. Science. 2010;327:1644–1648. doi: 10.1126/science.1184008. PubMed DOI PMC

Boulanger N, Lowenberger C, Volf P, Ursic R, Sigutova L, Sabatier L, Svobodova M, Beverley SM, Späth G, Brun R, Pesson B, Bulet P. Characterization of a defensin from the sand fly Phlebotomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major. Infect Immun. 2004;72:7140–7146. doi: 10.1128/IAI.72.12.7140-7146.2004. PubMed DOI PMC

Kumar S, Christophides GK, Cantera R, Charles B, Han YS, Meister S, Dimopoulos G, Kafatos FC, Barillas-Mury C. The role of reactive oxygen species on Plasmodium melanotic encapsulation in Anopheles gambiae. Proc Natl Acad Sci USA. 2003;100:14139–14144. doi: 10.1073/pnas.2036262100. PubMed DOI PMC

Zdobnov EM, von MC, Letunic I, Torrents D, Suyama M, Copley RR, Christophides GK, Thomasova D, Holt RA, Subramanian GM, Mueller HM, Dimopoulos G, Law JH, Wells MA, Birney E, Charlab R, Halpern AL, Kokoza E, Kraft CL, Lai Z, Lewis S, Louis C, Barillas-Mury C, Nusskern D, Rubin GM, Salzberg SL, Sutton GG, Topalis P, Wides R, Wincker P, Yandell M, Collins FH, Ribeiro J, Gelbart WM, Kafatos FC, Bork P. Comparative genome and proteome analysis of Anopheles gambiae and Drosophila melanogaster. Science. 2002;298:149–159. doi: 10.1126/science.1077061. PubMed DOI

Kanzok SM, Fechner A, Bauer H, Ulschmid JK, Muller HM, Botella-Munoz J, Schneuwly S, Schirmer R, Becker K. Substitution of the thioredoxin system for glutathione reductase in Drosophila melanogaster. Science. 2001;291:643–646. doi: 10.1126/science.291.5504.643. PubMed DOI

Dillon RJ, Ivens AC, Churcher C, Holroyd N, Quail MA, Rogers ME, Soares MB, Bonaldo MF, Casavant TL, Lehane MJ, Bates PA. Analysis of ESTs from Lutzomyia longipalpis sand flies and their contribution toward understanding the insect-parasite relationship. Genomics. 2006;88:831–840. doi: 10.1016/j.ygeno.2006.06.011. PubMed DOI PMC

Jaramillo-Gutierrez G, Rodrigues J, Ndikuyeze G, Povelones M, Molina-Cruz A, Barillas-Mury C. Mosquito immune responses and compatibility between Plasmodium parasites and anopheline mosquitoes. BMC Microbiol. 2009;9:154. doi: 10.1186/1471-2180-9-154. PubMed DOI PMC

Narasimhan S, Sukumaran B, Bozdogan U, Thomas V, Liang X, Deponte K, Marcantonio N, Koski RA, Anderson JF, Kantor F, Fikrig E. A tick antioxidant facilitates the Lyme disease agent's successful migration from the mammalian host to the arthropod vector. Cell Host Microbe. 2007;2:7–18. doi: 10.1016/j.chom.2007.06.001. PubMed DOI PMC

Molina-Cruz A, DeJong RJ, Charles B, Gupta L, Kumar S, Jaramillo-Gutierrez G, Barillas-Mury C. Reactive oxygen species modulate Anopheles gambiae immunity against bacteria and Plasmodium. J Biol Chem. 2008;283:3217–3223. PubMed

Sant'Anna MR, Alexander B, Bates PA, Dillon RJ. Gene silencing in phlebotomine sand flies: Xanthine dehydrogenase knock down by dsRNA microinjections. Insect Biochem Mol Biol. 2008;38:652–660. doi: 10.1016/j.ibmb.2008.03.012. PubMed DOI PMC

Dunkov B, Georgieva T. Insect iron binding proteins: Insights from the genomes. Insect Biochem Mol Biol. 2006;36:300–309. doi: 10.1016/j.ibmb.2006.01.007. PubMed DOI

Ramalho-Ortigao JM, Traub-Cseko YM. Molecular characterization of Llchit1, a midgut chitinase cDNA from the leishmaniasis vector Lutzomyia longipalpis. Insect Biochem Mol Biol. 2003;33:279–287. doi: 10.1016/S0965-1748(02)00209-6. PubMed DOI

Sadlova J, Volf P. Peritrophic matrix of Phlebotomus duboscqi and its kinetics during Leishmania major development. Cell Tissue Res. 2009;337:313–325. doi: 10.1007/s00441-009-0802-1. PubMed DOI PMC

Dinglasan RR, Devenport M, Florens L, Johnson JR, McHugh CA, Donnelly-Doman M, Carucci DJ, Yates JR, Jacobs-Lorena M. The Anopheles gambiae adult midgut peritrophic matrix proteome. Insect Biochem Mol Biol. 2009;39:125–134. doi: 10.1016/j.ibmb.2008.10.010. PubMed DOI PMC

Lehane MJ. Peritrophic matrix structure and function. Annu Rev Entomol. 1997. PubMed

Devenport M, Alvarenga PH, Shao L, Fujioka H, Bianconi ML, Oliveira PL, Jacobs-Lorena M. Identification of the Aedes aegypti peritrophic matrix protein AeIMUCI as a heme-binding protein. Biochemistry. 2006;45:9540–9549. doi: 10.1021/bi0605991. PubMed DOI

Anderson JM, Oliveira F, Kamhawi S, Mans BJ, Reynoso D, Seitz AE, Lawyer P, Garfield M, Pham M, Valenzuela JG. Comparative salivary gland transcriptomics of sandfly vectors of visceral leishmaniasis. BMC Genomics. 2006;7:52. doi: 10.1186/1471-2164-7-52. PubMed DOI PMC

Volf P, Volfova V. Establishment and maintenance of sand fly colonies. J Vector Ecol. 2011. p. 36. PubMed

Francischetti IM, Calvo E, Andersen JF, Pham VM, Favreau AJ, Barbian KD, Romero A, Valenzuela JG, Ribeiro JM. Insight into the Sialome of the Bed Bug, Cimex lectularius. J Proteome Res. 2010;9:3820–3831. doi: 10.1021/pr1000169. PubMed DOI PMC

Huang X, Madan A. CAP3: A DNA sequence assembly program. Genome Res. 1999;9:868–877. doi: 10.1101/gr.9.9.868. PubMed DOI PMC

Ewing B, Hillier L, Wendl MC, Green P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 1998;8:175–185. PubMed

Ewing B, Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998;8:186–194. PubMed

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–3402. doi: 10.1093/nar/25.17.3389. PubMed DOI PMC

Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25:25–29. doi: 10.1038/75556. PubMed DOI PMC

Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, Koonin EV, Krylov DM, Mazumder R, Mekhedov SL, Nikolskaya AN, Rao BS, Smirnov S, Sverdlov AV, Vasudevan S, Wolf YI, Yin JJ, Natale DA. The COG database: an updated version includes eukaryotes. BMC Bioinformatics. 2003;4:41. doi: 10.1186/1471-2105-4-41. Epub; 2003 Sep 11. PubMed DOI PMC

Schultz J, Copley RR, Doerks T, Ponting CP, Bork P. SMART: a web-based tool for the study of genetically mobile domains. Nucleic Acids Res. 2000;28:231–234. doi: 10.1093/nar/28.1.231. PubMed DOI PMC

Bateman A, Birney E, Durbin R, Eddy SR, Howe KL, Sonnhammer EL. The Pfam protein families database. Nucleic Acids Res. 2000;28:263–266. doi: 10.1093/nar/28.1.263. PubMed DOI PMC

Bendtsen JD, Nielsen H, von HG, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol. 2004;340:783–795. doi: 10.1016/j.jmb.2004.05.028. PubMed DOI

Sonnhammer EL, von HG, Krogh A. A hidden Markov model for predicting transmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol. 1998;6:175–182. PubMed

Julenius K, Molgaard A, Gupta R, Brunak S. Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites. Glycobiology. 2005;15:153–164. PubMed

Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23:2947–2948. doi: 10.1093/bioinformatics/btm404. PubMed DOI

Abascal F, Zardoya R, Posada D. ProtTest: selection of best-fit models of protein evolution. Bioinformatics. 2005;21:2104–2105. doi: 10.1093/bioinformatics/bti263. PubMed DOI

Schmidt HA, Strimmer K, Vingron M, von HA. TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics. 2002;18:502–504. doi: 10.1093/bioinformatics/18.3.502. PubMed DOI

Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 2007;24:1596–1599. doi: 10.1093/molbev/msm092. PubMed DOI

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