Castañeda, Oscar

Díaz Montilla, Ana Elizabeth

Gallego Sánchez, Gerardo J.

Suárez Baron, Harold

Tohme, Joseph M.

Vásquez, Wilson

Zapata, Patricia

CGSpace

Gerardo Gallego Sánchez¹, Patricia Zapata¹, Oscar Castañeda¹, Harold Suárez-Baron¹, Ana Elizabeth Díaz², Wilson Vásquez3 and Joe Tohme¹  DIAZ, A. E.; SOLIS, M. A. 2007. A New species and species distribution records of Neoleucinodes (Lepidoptera: Crambidae:Spilomelinae) from Colombia feeding on Solanum sp. Proceedings of the Entomological Society of  Washington. 109 (4):897–908.  FOLMER, O., M. BLACK, W. HOEH, R. LUTZ, AND R. VRIJENHOEK.1994. DNA primers for ampliﬁcation of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates.Mol. Mar. Biol. Biotechnol. 3: 294-297. GILBERTSON, R. L.; ROJAS, M.R.; RUSSELL, D.; MAXWELL, D. P. 1991. The use of the asymmetric polymerase chain reaction and DNA sequencing to determinate genetic variability among isolates of bean golden mosaic geminivirus in the Dominican Republic. J. Gen. Virol. 72: 2843-2848.  KATTAN, G. H.; FRANCO, P.; ROJAS, V.; MORALES, G. 2004. Biological diversification in a complex región: a spatial analysis of faunistic diversity and biogeography of the Andes of Colombia. Journal of Biogeography, 31, 1829-1839.   1 Agrobiodiversity  and  Biotechnology Project, International Center for Tropical Agriculture (CIAT).Cali, Colombia. 2 Colombian  Corporation  for  Agricultural Research  (CORPOICA), La Selva. Rionegro, Colombia; 3INIAP-CORPOINIAP. Ecuador.  . g.gallego@cgiar.org INTRODUCTION Use of DNA sequences for identification of possible biotypes of the fruit borer Neoleucinodes elegantalis (Lepidoptera: Crambidae), an important pest of Andean solanaceous fruits  MATERIALS AND METHODS In Colombia, Venezuela, Ecuador, Brazil and Honduras, the tomato borer, Neoleucinodes elegantalis, is the most important fruit-related plague of the Solanaceae family.  A suitable molecular characterization using a DNA barcoding system is necessary to clarify different issues inside the taxonomy of Neoleucinodes genus. Additionally, other DNA  sequences used for molecular identification and phylogenetics studies, can be implemented to obtain a better understanding of the genetic variability across different animal groups and allows to acquire a enhanced description of the population s genetic variation. The main objectives of this study are: 1. Evaluate the performance of DNA barcoding sequences (COI gen and 18S rDNA gene), in the genetic characterization of populations of N. elegantalis, collected in different wild and cultivated solanaceous plants in Colombia and Ecuador. 2. Determination of possible haplotypes related with each population belonging to this species. 3.Identification of geographical patterns associated with the distribution of this insect. DISCUSSION DNA extraction from 292 individuals collected in Colombia, Ecuador and Honduras  was performed using the GF – 1 Nucleic Acid Extraction Kit (GF1-100-Vivantis)  and the protocol proposed by  Gilbertson et al. (1991).  Amplifications were carried out using universal primers that flank the COI region, with COI-Forward (5’-GGTCAACAAATCATAAAGATATTGG-3’) and COI-Reverse (5’-TAAACTTCAGGGTGACCAAAAAATCA 3’) (Folmer et al. 1994).Additionaly a new set of primers was developed to amplify the 18S rDNA gene: Neol_CIAT-18S-01-For (5’-AAAGCGGGCTCAAAATGCTG-3’)  and Neol_CIAT-18S-05-Rev(5’-CGGTCCGAAGACCTCACTAA-3’). Purification of the PCR product was performed using the PCR Clean up system (Promega).  The sequences were obtained using an automatic sequencer ABI 3730 (Perkin Elmer/Applied Biosystem - Foster City, CA) from MACROGEN squencing service and then were assembled using the software Sequencher 4.6 (Gene Codes Corporation Ann Arbor; MI).  99 Sly bmh PM100 Sly bmh PM123 Squ NA129 Squ NA6 Sly bh PM88 Squ bh PM1 Squ bs PM126 Squ NA2 Squ bs PM110 Shi bmh PM7 Sly bh PM93 Squ bh PM105 Sly bmh PM122 Squ NA119 Sbe NA95 Sly bh PM125 Squ NA128 Squ NA4 Squ bs PM106 Sly bmh PM8 Squ bh PM124 Squ NA127 Squ NA97 Sly bh PM120 Sbe NA89 Squ bh PM91 Sbe bmh PM47 Squ bh MB3 Squ bs PM96 Squ bmh MB20 Sat bh PM23 Sbe bh PM130 Squ NA107 Squ bmh MB17 Squ bh MB24 Sbe bh PM90 Sly bh PM92 Squ bh PM121 Squ NA76 Shi bh PM94 Sbe bh PM5 Squ bmh PM22 Sly bh PM73 Shi bh PM77 Shi bh PM74 Shi bh PM80 Shi bh PM79 Shi bh PM78 Shi bh PM25 Scr bh PM50 Squ bh PM26 Scr bh PM48 Sly bh PM45 Squ bh PM27 Sly bh PM18 Squ bh PM41 Sly bh PM29 Sly bh PM49 Squ bh PM31 Squ bh PM28 Sly bh PM40 Sly bh PM37 Squ bh MB39 Sly bh PM35 Squ bh MB34 Squ bh PM36 Squ bh MB32 Squ bh PM53 Squ bh PM67 Sly bmh PM87 Squ bmh PM68 Sbe bmh PM71 Squ bmh PM75 Sbe bmh PM85 Sbe bmh PM55 Sbe bmh PM63 Sly bmh PM70 Squ bmh PM58 Sly bmh PM82 Squ bh PM62 Sbe bmh PM61 Sly bmh PM64 Sly bmh PM59 Squ bmh PM51 Sly bmh PM83 Sly bh PM52 Sly bmh PM57 Sbe bmh PM56 Squ bmh PM60 Sbe bmh MB81 Squ bh PM72 Shi bh PM54 Squ bmh PM86 Squ bmh PM43 Sla bh PMDifferent bioinformatic tools were used for alignment and verification of quality  (Sequencher 4.6, BioEdit7.0.9.0). The nucleotide composition for each sample, genetic distances and heterogeneity among sequences (MEGA5). The haploypes determination was obtained from DnaSP v5.  Additionally we performed an analysis to establish the geographic distribution for each sample was determined, using the software ArcGIS – ArcMap vs.10 (ERSI 1999 – 2008). The genetic distances between sequences were calculated using Neighbor-Joining/UPGMA algorithm implemented in MEGA 5. These distances yield 5 principal groups, of which 2 belong to Colombia, the other groups represents to Ecuador, Honduras and Costa Rica.   Divergence between groups was very high and the greatest were found in one specific group from Colombia, which was distributed all along the Western Cordillera, and was also apparently geographically isolated from the rest of the populations of  N. elegantalis.   COI 18S rDNA  a. Geographical distribution of most common N. elegantalis haplotypes in Colombia, b. Clasification of the 5 sub-regions proposed by Kattan et al., (2004), also refered to by the author as faunistic zones. Cauca Valley, Central Cordillera and Magdalena Valley are the three sub-regions where all Colombian individuals come from.  DNA Barcoding was an accurate tool for the identification of haplotypes as well as discrimination of species (N. silvane) reported previously by Díaz and Solís (2007) using geometric morphology. The number of haplotypes obtained revealed a possible role of biogeographic isolation between valleys and of possible human pressure through the use of pesticides inducing divergent selection in N. elegantalis. The NJ analysis shows a wide distribution of the species along the Magdalena valley watershed, this sub-region is located between the Central and Eastern Cordilleras. The distribution of N. elegantalis in this region could explain the wide range of altitudinal adaptation of the species that could facilitate dispersal. With regard to the Cauca Valley, diversity centers on the southwestern slope of the Central Cordillera and on the East of Western Cordillera. The classification of regions proposed by Kattan et al (2004), are highly correlated to the grouping of haplotypes recovered in the species. The evaluation of  sequences of Cytocrome c oxidase I (COI) and18S rDNA gene,  as a  one single sequences reveals a powerful tool in haplotypes characterization, showing a strong differentiation in relation with the geographic origin of each sample.   The analysis of the COI showed good sensitivity, achieving an initial differentiation of 49 possible haplotypes, with some association to specific life zones, but without apparent relation to the host type.    Genetic distances were inferred using the Maximum Likelihood (ML) method and including the evolutive model GTR+G. The sequences used to construct the dendrogram corresponds to Cytocrome c oxidase I (COI) and18S rDNA sequences respectively.  The evaluation of both sequences in a combined way allows a better characterization of samples according their geographical origin. Besides it was possible to improve the resolution in term of genetic differentiation into samples distributed across Colombia and Ecuador. This distribution was associated with the faunistic zones proposed by Kattan et al (2004), for the specific case of Colombia.  a b   • The DNA Barcoding tool shows high sensitivity in N. elegantalis haplotype identification, and to correlation with the sub-regions of colombia which suggest at least 3 different geographic groups, related to both biogeographic separation, and human intervention through the use of pesticides.  • The evaluation of  sequences of Cytocrome c oxidase I (COI) and18S rDNA gene,  as a  one single sequences reveals a powerful tool for N. elegantalis haplotypes characterization • This is the first genetic analysis of N. elegantalis and the first attempt to obtain a molecular characterization of the species.  • Genetic differentiation could mean that there is partial reproductive isolation in N. elegantalis; further research could center on resolving this issue.  • Through this methodology we confirm the existance of a new species of Neoleucinodes genera (N. silvanie) previously proposed by Díaz and Solís (2007) with the use of morphological characters.  • Thus, for N. elegantalis,both mitochondrialDNAdifferentiation and morphological divergence provide evidence for the existence of populations that are going through ecological speciation CONCLUSIONS Many thanks to the Regional Fund for Agricultural Technology (FONTAGRO) for funding this study ACKNOWLEDGMENTS BIBLIOGRAPHY RESULTS Additionally, our possible new species samples are clustered with individuals included in the group associated with samples from Euleucinodes genus. 

Use of DNA sequences for identification of possible biotypes of the fruit borer Neoleucinodes elegantalis (Lepidoptera: Crambidae), an important pest of Andean solanaceous fruits

https://cgspace.cgiar.org/bitstream/10568/67052/1/Use%20of%20DNA%20sequences%20for%20identification%20of%20possible%20biotypes%20of%20the%20fruit%20borer.pdf

Use of DNA sequences for identification of possible biotypes of the fruit borer Neoleucinodes elegantalis (Lepidoptera: Crambidae), an important pest of Andean solanaceous fruits

Abstract

Similar works

Full text

Available Versions

CGSpace