AZEREDO-ESPIN, A. M. L.

BERGAMO, L. W.

MASTRANGELO, T.

MORAIS, E. G. F. de

PAULO, D. F.

SILVA, M.

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TÍTULO DO RESUMODaniel F. Paulo1; Thiago Mastrangelo1; Luana W. Bergamo1; Elisangela G. F. de Moraes2;  Márcio Silva3 & Ana M. L. Azeredo-Espin1DETECTION OF Helicoverpa armigera POPULATIONS IN NORTH AND NORTHEAST OF BRAZIL1Centro de Biologia Molecular e Engenharia Genética (CBMEG), UNICAMP, Campinas-SP2EMBRAPA Roraima, Boa Vista-RR3Universidade Estadual do Piauí (UESPI), Uruçuí-PIdaniel.f.paulo@cbmeg.unicamp.brINTRODUCTION MATERIAL and METHODSBrazilian producers have reported reduced yields up to 35% on major crops during the 2012/2013 harvest due to the recently introduced moth Helicoverpa armigera (Hübner). The economic losses reached US$ 1 billion only in Western Bahia, triggering a phytosanitary crisis. The deficiencies in existing taxonomic keys to deal with the morphologically indistinct larvae of H. armigera and the native Helicoverpa zea (Boddie) have constraint the detection of new incursions of this heliothine invader. From March to July 2013, heliothine larvae were found infesting crops in Roraima, Piauí and  Bahia states. The aim of this study was, therefore, to identify these larvae through sequences of  the mitochondrial cytochrome c oxidase subunit I Larvae were collected from host plants in the states of Roraima, Piauí andBahia, and preserved in 100% ethanol at -20 ºC. Total DNA was extracted from thelast three abdominal segments of the larvae through the phenol: chloroformmethod. The partial sequences of the COI gene were amplified by using the primers(COIF and COIR) and PCR conditions described byLi et al.(2011). Amplicons were purified with theIllustraTMGFXTM kit and sequenced by theABI3730xl DNAAnalyzer sequencer. The65 COI fragments from the heliothinespecimens from Roraima (n=14),Piauí (n=39), and Bahia (n=12) wereused to perform the phylogenetic analysesalong with sequences of 3 Spodoptera frugiperda,one Heliothis virescens,gene (COI).RESULTS and DISCUSSION  CONCLUSION Figure 1. Neighbor-Joining strict consensus tree (topology under p-distance model) inferred using COIcomplete dataset for Heliothine species. The 65 specimens from Roraima (RR01-RR14), Piauí (PI01-PI39) and Bahia (BA01-BA12) are in red. Sequences of Spodoptera frugiperda, Heliothis virenses andHelicoverpa punctigera were used as outgroups. Numbers above branches refer to Neighbor-joining(NJ) bootstrap (BS) proportions among 5.000 replicates, while numbers below branches refers toBayesian inference (BI) posterior probability (PP) and Maximum-likelihood (ML) BS proportionsamong 1.000 replicates. The four analyses gave nearly identical tree topologies, and node supportsvalues below 70% (BS) and/or 0.7 (PP) were not recorded in the tree (-).one Helicoverpa punctigera,one Helicoverpa gelotopoeon,one Helicoverpa hawaiiensis,3 Helicoverpa assulta, 5 Helicoverpazea, and the 16 H. armigerahaplotypes reported by Li et al.(2011)(arm01 – arm16). Neighbor-joining (NJ)analysis was calculated in MEGA 5.1 softwarewith Kimura two-parameters (K2P) and uncorrected sequence divergences (p-distances) models. The Bayesian inference (BI) and Maximum-likelihood (ML) methods werealso performed.All specimens yielded high-quality DNAand were successfully sequenced for the COIfragment (658 bp). The four phylogeneticanalyses (NJ under p-distance and K2P models,BI and ML under MrAIC 1.4.4 favored substitutionmodel) gave nearly identical tree topologies. The65 heliothine specimens fromRoraima, Piauí and Bahia joinedby more than 95% BS and0.9 PP values, forming a monophyleticgroup with the 16 H. armigera sequencesreported by Li et al.(2011). The H. armigeragroup formed a robustly supportedmonophyletic clade with H.zea, andthis same phylogenetic pattern had beenThe information content of the COI fragment selected in this study was sufficient toenable recognition of the divergences among the lepidopterans analyzed. The mtDNACOI phylogeny strongly supported that the 65 heliothine larvae from Roraima, Piauíand Bahia are conspecific with H. armigera, proving that this exotic pest is widelyspread in North and Northeast of Brazil.observed in previous studies, which havesuggested that the American H. zea populations were established via founder event fromH. armigera (or from their joint common ancestor) no more than 1.5 million years ago,what is further supported by the high similar morphology and the possibility of matingcompatibility between the two species. The other defined species (S. frugiperda, Heliothisvirescens, and the other 5 Helicoverpa species) were recovered in distinct positions or cladeson strongly supported branches.

Detection of Helicoverpa armigera populations in North and Northeast of Brazil.

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