Sex Identification of South American Parrots (Psittacidae, Aves
Transcrição
Sex Identification of South American Parrots (Psittacidae, Aves
516 ShortCommunications andCommentaries DAVIES,M. B., J. AUSTIN, AND D. A. PARTRIDGE.1991. VitaminC: Its chemistryandbiochemistry. Royal Society of Chemistry, Cambridge, United Kingdom. DIAMOND,J. M. 1986. Why do disusedproteinsbecomegeneticallylost or repressed? Nature 351: [Auk, Vol. 114 JENNESS,R., E. C. BIRNEY,AND K. L. AYAZ. 1980. Variationof L-gulonolactone oxidaseactivity in placentalmammals.ComparativeBiochemistry and Physiology67B:195-204. MADDISON, W. P., AND D. R. MADDISON. 1992. MacClade (version3): Analysis of phylogeny and character evolution. Sinauer Associates, 565-566. ELLIOT,O., N.J. YESS,AND D. M. HEGSTED.1966. Bio- Boston. synthesisof ascorbicacid in the tree shrew and PAULING, L. 1970. Vitamin C and the common cold. the slow loris. Nature W. H. Freemanand Company,San Francisco. PIANKA,E. R. 1994. Evolutionaryecology.Harper 212:739-740. ENGLARD,S., AND S. SEIFTER.1986. The biochemical functions of ascorbic acid. Annual Nutrition 6:365-406. Review of FITCH,W. 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Pages432-448 in Modern nutrition in health and disease,vol. 1 (M. E. Shils, J. A. Olson, and M. Shike, Eds.). Lea and Febinger,New York. University Press,New Haven, Connecticut. Received 3 September 1996,accepted 5 February1997. Associate Editor:M. E. Murphy TheAuk 114(3):516-520,1997 Sex Identification of South American Parrots(Psittacidae,Aves) Using the Human Minisatellite Probe 33.15 CRISTINAY. MIYAKI,• J. MAURICIOB. DUARTE,2 RENATOCAPARROZ, •'2 ADAUTO L. V. NUNES,3 AND ANITA WAJNTALTM •Departamento deBiologia, Universidade deSaoPaulo,C.P.11,461,CEP05422-970,SaoPaulo,SP,Brazil; 2Departamento deMelhoramento Gen•ticoAnimal,FCAVJ,UNESP, RodoviaCarlosTonanniKm5--Jaboticabal, CEP 014870-000,St•oPaulo,SP,Brazil;and 3Parque Zooldgico deSorocaba, Sorocaba, SP,Brazil Many speciesof SouthAmericanparrotsare endangered,and captivebreeding has becomea standard procedure for speciesconservation.Because most South American parrots are not sexually dimorphic, an efficientmeansof determining the sex of individuals is an important tool in establishing and maintaining a viable breeding population in captivity. DNA fingerprinting(Jeffreyset al. 1985)hasbeen Addresscorrespondence to this author.E-mail: [email protected] appliedin a varietyof wild species,includingbirds (Burke and Bruford 1987, Wetton et al. 1987). It was usedto monitorgeneticvariabilityin captivePuerto Rican Parrots (Amazona vittata; Brock and White 1992)and to establishpaternityin endangeredspecies(Math6 et al. 1993).Recently,we used DNA fin- gerprintingto identifythe sexof Peach-fronted (Aratingaaurea)and Golden (Guaruba[Aratinga]guarouba)parakeets(Miyaki et al. 1992)and suggested that fingerprintingalsocouldbe usedto determine sex in other psittacines(Miyaki et al. 1993, 1995). Here, we presentresultson sex determinationusing the human minisatelliteprobe33.15 (Jeffreyset al. July1997] Short Communications andCommentaries 517 TABLE 1. Patternof intensebandsin previously sexedandunsexed parrots.F andM arenumberof females and males;N and Nb are number of individuals and numberwith intensebands,respectively. Sexed Species Method a Unsexed F M N Nb Amazona aestivaaestiva 5 8 k Bands b - 10 0 Total 23 Amazona aestiva xanthopteryx 1 3 k - 0 2 1 7 0 0 0 0 4 6 2 11 Amazona amazonica Amazona autumnalis diadema Amazona brasiliensis 2 1 2 2 0 2 k k k, 1 Amazona dufresniana rhodocorytha Amazona farinosa Amazona festiva Amazona ochrocephala xantholaema Amazona pretrei 0 0 0 1 3 2 0 2 2 1 3 4 k k b d - 4 3 4 5 4 0 0 0 0 0 4 5 6 7 10 Amazona xanthops Anodorhynchus hyacinthinus Anodorhynchus leari 1 3 1 0 5 1 k k k, 1 + + 4 55 1 0 32 1 5 63 3 Ara ararauna 3 2 k, b + 12 8 17 Arachloroptera 2 5 k, b + 15 9 7 4 4 4 22 Ara maracana Ara nobilis 4 2 1 4 k k + + 2 11 0 3 7 17 Aratinga acuticaudatta Aratinga aurea Aratinga leucophthalmus Aratinga mitrata Aratinga solstitialis auricapilla Aratinga solstitialis jandaya Cyanopsitta spixii Deroptyus accipitrinus Guaruba guarouba Nandayus nenday Pionites leucogaster Pionopsitta pileata 1 2 1 0 0 0 1 2 1 0 0 0 k k b + + + 0 32 10 2 8 4 14 5 1 2 4 2 36 12 2 8 4 3 1 3 2 1 1 4 1 3 0 1 1 k b k, b b k d + + + - 0 0 19 21 4 6 9 10 0 0 7 2 25 23 6 8 Pyrrhura egregia Pyrrhura frontalis Pyrrhura picta 1 1 1 0 1 1 b b b - 1 2 3 0 0 0 2 4 5 Amazona vinacea Ara auricollis Ara macao Ara manilata Pionusmenstruus Triclaria malachitacea 2 2 2 2 1 1 5 1 2 1 k, b k k k b d - + + + - - 6 1 7 0 0 1 0 - 12 4 16 10 2 k, karyotypeanalysis; 1,laparoscopy; b, breeding behavior; d, sexualdimorphism. Female-specific bands:-, absent;+, present. or HaelII (for the 1985)in 36 speciesbelongingto 13 generaof South tion enzymeMboI (for Amazona) othergenera).Fragmentswereseparatedby electroMethods.--Bloodsamples were collected from phoresisthrougha 30-cmlong 1% horizontalagawas stoppedwhen the birds belongingto aviculturistsand officialestab- rose gel. Electrophoresis lishments in Brazil. For some individuals, sex was 2-kilobase(kb) markerbandhadmigratedto thebotDNA fragmentswere determinedby karyotyping,laparoscopy, or breed- tomof thegel.Thefractionated ing behavior.Wheneverpossible,growingfeathers transferredontoa nylonmembraneby standardcapblotting(Sambrook et al. 1989). (ca.25 daysold) werecollectedfrombirdswhosesex illary Southern The membranewashybridizedwith minisatellite wasunknownand processed for karyotypeanalysis. et al. 1985),whichwaslabeled Chromosomepreparationand analysisfollowed 33.15probe(Jeffreys dCTP or [ct-32P] dATP.Pre-hybridization Duarte and Caparroz(1995).The speciesand num- with [ct-32P] ber of individuals studied are shown in Table 1. in 0.263M Na2HPO4,1 mM EDTA, 7% SDS,and 1% The protocolsused to obtain multilocusfinger- BSA at 65øClastedfor 4 h, and the probewas added printsfollowedBrufordet al. (1992).Foreachbird, 5 and left overnight at 65øC. The membrane was •g of genomicDNA were digestedwith the restric- washed in 0.25 M Na2HPO4,1% SDS, 2XSSC, 0.1% American parrots. 518 ShortCommunications andCommentaries A B C D E F G kbFFMMMFFFFMMFFMFFMFFMMFFMM H kb FM [Auk,Vol. 114 I F F MMM 23- 4.4- FIG. 1. ParrotDNA hybridizedwith human minisatelliteprobe33.15.Female-specific bandsare shown with arrows. (A) Ara auricollLs, (B) Am chloroptera, (C) Ara macao,(D) Ara manilata,(E) Am maracana, (F) Ara nobills,(G) Anodorhynchus hyacinthinus, (H) Anodorhynchus leari,(I) Cyanopsitta spixii.F = female,M = male. TABLE 2. Molecular size of the sex-linked bands in SouthAmericanparrots. Number Species Molecular of bands size (kb) Anodorhynchus hyacinthinus Anodorhynchus leari 3 2 3.1; 5.1; 5.2 3.1; 18.5 Ara ararauna Ara auricollis 2 4 1.9; 2.5 2.7; 3.7; 3.9; 4.8 Ara chloroptera 4 2.7; 3.7; 4.0; 4.1 Ara Ara Ara Ara 4 3 3 3 2.5; 2.7; 2.7; 2.5; 2 3 2 3 3.7; 7.6 2.9; 3.6; 4.6 3; 7.6 2.9; 3; 10.5 3 4.2; 5.0; 6.0 3 2 2 3 4.1; 5.2; 5.8 3.1; 3.5 2.8; 5.7 3.9; 4.8; 6.0 macao manilata maracana nobilLs Amtingaaurea Aratingaacuticaudatta Aratingaleucophthalmus Aratingareitrata Aratingasolstitialis auricapilla Aratingasolstitialis jandaya Cyanopsitta spixii Guarubaguarouba Nandayusnenday 4.0; 3.5; 3.9; 4.7; 4.1; 4.3 4.0 4.6 11.5 SDS and in IxSSC, 0.1% SDS at 65øC. The filter was then autoradiographedat -70øC using Kodak RX film with one or two intensifyingscreens. Results.--Apatternof two to four intensesex-specific bandswas detectedwith the 33.15 probe in all femalesstudiedin the generaAra and Aratingaand alsoin femalesof Anodorhynchus hyacinthinus, A. leari, Cyanopsitta spixiLGuarubaguarouba,and Nandayus nenday(see Fig. 1). These intense bands also were presentin somepreviouslyunsexedindividualsof Aratingasolstitialis auricapilla, A. s.jandaya, andA. mitrata(Table1). Eachspeciesshoweduniquepatterns of female-linkedbandsaccordingto their molecular size(Table2); thesespecies-specific profilesarea potential tool for speciesidentification.Female-linked bandsalsowere observedin long-tailedspecieseven when otherrestrictionenzymeswereused(datanot shown). Sex-linked bands were absent in known fe- malesof eight speciesof Amazonaand of Pionusmenstruus, Deroptyusaccipitrinus,Pionites leucogaster, Pionopsitta pileata,Pyrrhuraegregia, P.frontalis,P.picta, and Triclaria malachitacea. We used MboI for members of Amazonabecausethis enzyme produced more polymorphicDNA fingerprint profiles;even when July1997] ShortCommunications andCommentaries we used HaeIII, no sex-linkedfragmentswere obDiscussion.--Until recently,only threemethodsof were available for birds: fecal hor- moneassay,chromosome analysis,and laparoscopy. Hormoneanalysesvary with reproductivecondition and thus are age and seasondependent.Chromosomeanalysisis reliablebut time consuming,andin many occasionsappropriatemetaphasecellscannot be found for analysiswithout repeatedattempts. Birdshaveto behandledtwiceforkaryotyping,once for removinga sampleof feathersand a secondtime for collectingthe growingfeatherpulp. Because laparoscopyis a surgicalprocedure,birds are exposed to anesthesia and to the risksof surgery. Recently,severalDNA techniqueshave been developed for sex identification in birds (de Kloet and de Kloet 1992,Griffithsand Tiwari 1993,May et al. 1993).Griffiths and Tiwari (1995) determined the sex of the last Spix'sMacaw (Cyanopsitta spixii) from shed feathers that were collected in the wild and used as a sourceof DNA for PCR amplification.In our applicationof DNA fingerprintingin South Americanparrots,we identified female-specific intensebandsin somespeciesusing the human minisatelliteprobe33.15(Miyaki et al. 1992,1993).Other authors also have detected sex-specificbands in DNA fingerprintingof birds usingprobe33.15 (Rabenoldet al. 1991,Longmireet al. 1992)and probe 33.6 (Graveset al. 1993).Althoughprobe33.15could not be used to identify the sexof all the specieswe studied,it was invaluablefor sexingmacaws(Ara, Anodorhynchus, andCyanopsitta) andconures(Aratinga and Nandayus). Thesegenerabelongto the "long, point-tailed"groupand areconsidered to be closely related. Our method tern of a few intense bands detected in some unsexed was restricted to females. individuals served (data not shown). sex identification 519 Little is known about the phylogeneticrelationshipsof psittacines.The availabledata are basedon chromosomal evolution (Valentine 1990, Christidis et al. 1991),albumin(Sibley1960),andthecytochromeb gene(Birt et al. 1992,Leetonet al. 1994).Basedon karyotypecorrelations, Valentine(1990)proposedan early separationof Amazonafrom other genera. Based on habitat exploration,Mont6n (1977) suggestedthat the short-tailedand the long-tailedbirds havebehavioraldifferences.The presenceof w-chromosome-linked minisatellitesequences in mostlongand sharp-tailed New World psittacines,and their absencein the short-tailedspecies,provide support for the separateevolutionof thesetwo groups.Preliminaryanalysisofmitochondrial gene(12Sand16S rDNAs and cytochrome-b)sequences of nine species of Brazilianparrotsalsosupportsthe separationof the long-and short-tailedspecies(Miyaki 1996). Acknowledgments.--This work was fundedby FAPESP,CNPq, and CAPES(Brazil). We thank T. Burke for a visit to his lab; O. Hanotte and C. E M. Menck for their constantsupport;P.F. Flechafor invaluable discussion;and L.A. Labruna (ParqueEco16gicodo Tiet•, SP), A. L. V. Nunes (SorocabaZoo), N. Kawall, L. Maluf, A. Marra, A. Vertematti, C. Isoldi, and M. Silva (aviculturists,Brazil), L. Sanfilippo(SgoPaulo Zoo), and M. I. Bampi (IBAMA) for parrot blood samples.C. Y. M. hasa FundoBUNKA/90 prize.The Jeffreys'probes33.6 and 33.15are the subjectof patent No. GBA 2166445and worldwide patentsfor commercialdiagnosticuse.We alsothank Associate Editor Allan Bakerand two anonymousreviewers for commentsand editorialsuggestions. could not be used to sex the LITERATURE short-tailedparrots in the genusAmazona,and our CITED datasuggestthat it alsowasnot adequatefor sexing BIRT, t. P., V. L. FRIESEN,J. M. GREEN,W. A. MONother short-tailedspeciessuch as Pionusmenstruus, Pionites leucogaster, and Pionopsitta pileata(whichhas sexuallydimorphicplumage),as well as the "long, wide-tailed" Deroptyusaccipitrinusand Triclariama- lachitacea (the latter has sexuallydimorphicplumage). The absenceof female-linked bands was not dueto the factthata differentrestrictionenzymewas applied. No sex-specificpattern was detectedin the three speciesof Pyrrhurawe studied,eachof which has a long, sharp-pointedtail. TEVECCHI,AND W. S. DAVIDSON.1992. 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SCHODDE. 1991. 33.15.However,the agreementbetweenthe DNA resuitsand the cytogenetic datain all speciesfor which both studieswere performed suggeststhat the pat- Chromosomal evolution in parrots, 1orikeets and cockatoos(Aves:Psittaciformes).Hereditas 114:47-56. 520 ShortCommunications andCommentaries DE KLOET, D. H., AND $. R. r•E KLOET. 1992. Molec- ular determinationof the sexof parrots.Focus 14:106-108. [Auk,Vol. 114 tation, Universidade de Silo Paulo, Silo Paulo, Brazil. MIYAKI, C. Y., O. HANOTTE, g. WAJNTAL, AND t. DUARTE, J.M. B, ANDg. CAPARROZ. 1995. Cytotaxonomicanalysisof Brazilianspecies of thegenus Amazona (Psittacidae, Aves) and confirmation of the genus Salvatoria(Ribeiro, 1920). Brazilian Journal of Genetics 18:623-628. GRAVES,J., J. ORTEGA-gUANO,AND P. J. g. SLATER. 1993. Sexratioof chicksin the ShagPhalacrocorax aristotelisdetermined by a female-specific band in DNA fingerprinting.Ibis 135:470-472. GRIFFITHS,g., AND B. TIWARI. 1993. The isolation of BURICE. 1992. 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