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Global and local effects of pigmentation in Bicyclus anynana

Marcelino, Ana Carina Fernandes, 1986-
Fonte: Universidade de Lisboa Publicador: Universidade de Lisboa
Tipo: Dissertação de Mestrado
Publicado em //2010 ENG
Relevância na Pesquisa
47.68%
Tese de mestrado. Biologia (Biologia Evolutiva e do Desenvolvimento). Universidade de Lisboa, Faculdade de Ciências, 2010; The astounding diversity, ecological relevance and insights into the underlying development, make butterfly wing pigmentation an excellent model for evo-devo. These two-dimensional colour patterns depend on two sequential phases during pupal development: transcription factors first give patterning information to cells, and effector enzymes then produce specific colour pigments that are deposited in the developing scale-cells that will decorate the adult wings. The sequence of pigment deposition occurs in a stereotypical order, and the time of scale maturation is strongly correlated with scale colour. However, the relationship between colour and the time of maturation of scale-cells remains to be understood. We use two spontaneous mutants of a butterfly lab model to explore this relationship. Melanine mutants, with a global change darken the whole body and Fred mutants with a local. In this project we characterize the developmental timing of pigmentation in these mutants. We use ISH and semi-quantitative PCR to determine the genetic expression of candidate genes for pigmentation. Our results show that in melanine mutants pigment deposition occurs as in “wild-type” butterflies...

The Genetics and Development of an Eyespot Pattern in the Butterfly Bicyclus Anynana: Response to Selection for Eyespot Shape

Monteiro, A.; Brakefield, P. M.; French, V.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1997 EN
Relevância na Pesquisa
47.34%
The normally circular eyespots on the wing of the butterfly Bicyclus anynana were selected to become elliptical in two divergent lines, with antero-posterior elongation of the eyespots in one line and proximodistal elongation in the other. Selection was continued for nine generations, and symmetrical realized heritabilities of ~15% were achieved initially. The elliptical eyespot shapes characteristic of each line were still produced when the signaling center of the eyespot (the focus) was surgically rotated by 90 or 180° or when an eyespot was induced ectopically by localized damage. We conclude that selection changed general properties of the epidermis that responds to signals emanating from the eyespot focus but did not affect the mechanism of focal signaling.

Female choice depends on size but not symmetry of dorsal eyespots in the butterfly Bicyclus anynana.

Breuker, Casper J; Brakefield, Paul M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 22/06/2002 EN
Relevância na Pesquisa
47.34%
The eyespots on the ventral wings of Bicyclus anynana butterflies are exposed when at rest and interact with predators. Those on the dorsal surface are not exposed in this way, and may be involved in courtship and mate choice. In this study, we examined whether the size and fluctuating asymmetry (FA) of dorsal eyespots are reliable signals of male quality. High developmental stability is considered to result in low FA, and to be associated with high quality. Individuals of high quality are predicted to produce sexually selected traits that are large and symmetrical, at a relatively low cost. In this study, we manipulated eyespot development to uncouple eyespot size and FA in order to examine their independent roles in signalling to the female. Individual females in cages were given the choice between two or three males differing in eyespot traits. The results indicate that although size per se of the eyespots is used as a signal, FA and wing size are not. We discuss the use of FA in studies of sexual selection and aspects of sexual selection on dorsal eyespot size.

Does predation maintain eyespot plasticity in Bicyclus anynana?

Lyytinen, Anne; Brakefield, Paul M.; Lindström, Leena; Mappes, Johanna
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 07/02/2004 EN
Relevância na Pesquisa
47.51%
The butterfly Bicyclus anynana exhibits phenotypic plasticity involving the wet-season phenotype, which possesses marginal eyespots on the ventral surface of the wings, and the dry-season form, which lacks these eyespots. We examined the adaptive value of phenotypic plasticity of B. anynana in relation to the defence mechanisms of crypsis and deflection. We assessed the visibility differences between spotless and spotted butterflies against backgrounds of brown (dry season) or green (wet season) leaves. Spotless butterflies were highly cryptic and less predated by adult bird predators than were spotted ones when presented against brown leaf litter. However, the advantage of crypsis disappeared in the wet-season habitat as both forms were equally visible. In later experiments, naive birds presented with resting butterflies in the wet-season habitat tended to learn more rapidly to capture spotless butterflies, suggesting a slight selective advantage of possessing eyespots. Moreover, marginal eyespots increased significantly the escape probability of butterflies that were attacked by naive birds compared to those attacked by adult birds, although there were no differences in prey capture success within naive predators. Our results show that natural selection acts against eyespots in the dry season...

Germline transformation of the butterfly Bicyclus anynana.

Marcus, Jeffrey M; Ramos, Diane M; Monteiro, Antónia
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 07/08/2004 EN
Relevância na Pesquisa
47.34%
Ecological and evolutionary theory has frequently been inspired by the diversity of colour patterns on the wings of butterflies. More recently, these varied patterns have also become model systems for studying the evolution of developmental mechanisms. A technique that will facilitate our understanding of butterfly colour-pattern development is germline transformation. Germline transformation permits functional tests of candidate gene products and of cis-regulatory regions, and provides a means of generating new colour-pattern mutants by insertional mutagenesis. We report the successful transformation of the African satyrid butterfly Bicyclus anynana with two different transposable element vectors, Hermes and piggyBac, each carrying EGFP coding sequences driven by the 3XP3 synthetic enhancer that drives gene expression in the eyes. Candidate lines identified by screening for EGFP in adult eyes were later confirmed by PCR amplification of a fragment of the EGFP coding sequence from genomic DNA. Flanking DNA surrounding the insertions was amplified by inverse PCR and sequenced. Transformation rates were 5% for piggyBac and 10.2% for Hermes. Ultimately, the new data generated by these techniques may permit an integrated understanding of the developmental genetics of colour-pattern formation and of the ecological and evolutionary processes in which these patterns play a role.

The use of chemical and visual cues in female choice in the butterfly Bicyclus anynana

Costanzo, Katie; Monteiro, Antónia
Fonte: The Royal Society Publicador: The Royal Society
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
47.34%
Investigating the relative importance of multiple cues for mate choice within a species may highlight possible mechanisms that led to the diversification of closely related species in the past. Here, we investigate the importance of close-range pheromones produced by male Bicyclus anynana butterflies and determine the relative importance of these chemical cues versus visual cues in sexual selection by female choice. We first blocked putative androconial organs on the fore- and hindwings of males, while also manipulating the ability of females to perceive chemical signals via their antenna. We found that male chemical signals were emitted by both fore- and hindwing pairs and that they play an important role in female choice. We subsequently tested the relative importance of these chemical cues versus visual cues, previously identified for this species, and found that they play an equally important role in female choice in our laboratory setting. In addition, females will mate with males with only one signal present and blocking both androconial organs on males seems to interfere with male to male recognition. We discuss the possible functions of these signals and how this bimodal system may be used in intra- and interspecific mate evaluation.

A Gene-Based Linkage Map for Bicyclus anynana Butterflies Allows for a Comprehensive Analysis of Synteny with the Lepidopteran Reference Genome

Beldade, Patrícia; Saenko, Suzanne V.; Pul, Nicolien; Long, Anthony D.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
47.51%
Lepidopterans (butterflies and moths) are a rich and diverse order of insects, which, despite their economic impact and unusual biological properties, are relatively underrepresented in terms of genomic resources. The genome of the silkworm Bombyx mori has been fully sequenced, but comparative lepidopteran genomics has been hampered by the scarcity of information for other species. This is especially striking for butterflies, even though they have diverse and derived phenotypes (such as color vision and wing color patterns) and are considered prime models for the evolutionary and developmental analysis of ecologically relevant, complex traits. We focus on Bicyclus anynana butterflies, a laboratory system for studying the diversification of novelties and serially repeated traits. With a panel of 12 small families and a biphasic mapping approach, we first assigned 508 expressed genes to segregation groups and then ordered 297 of them within individual linkage groups. We also coarsely mapped seven color pattern loci. This is the richest gene-based map available for any butterfly species and allowed for a broad-coverage analysis of synteny with the lepidopteran reference genome. Based on 462 pairs of mapped orthologous markers in Bi. anynana and Bo. mori...

The Genetic, Morphological, and Physiological Characterization of a Dark Larval Cuticle Mutation in the Butterfly, Bicyclus anynana

Bear, Ashley; Simons, Ariel; Westerman, Erica; Monteiro, Antónia
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 14/07/2010 EN
Relevância na Pesquisa
47.34%
Studies on insect melanism have greatly contributed to our understanding of natural selection and the ultimate factors influencing the evolution of darkly pigmented phenotypes. Research on several species of melanic lepidopteran larvae have found that low levels of circulating juvenile hormone (JH) titers are associated with a melanic phenotype, suggesting that genetic changes in the JH biosynthetic pathway give rise to increased deposition of melanin granules in the cuticle in this group. But does melanism arise through different molecular mechanisms in different species? The present study reports on a Bicyclus anynana (Lepidoptera: Nymphalidae) dark larvae single locus mutation, in which larvae exhibit a darker cuticle relative to wild type. Unlike other lepidopteran melanic larvae mutations, this one is autosomal recessive and does not appear to involve a deficiency in JH titers. Unlike JH deficiency mutants, dark larvae mutants display similar growth rates and sexual behaviors as wild type, and topical application of a JH analogue failed to rescue the wild type cuticular coloration. Finally, transmission electron microscopy showed that sclerotization or deposition of diffuse melanin, rather than deposition of melanin granules, produces the dark coloration found in the cuticle of this species. We conclude that different molecular mechanisms underlie larval melanism in different species of Lepidoptera.

In Vivo Electroporation of DNA into the Wing Epidermis of the Butterfly, Bicyclus anynana

Golden, Kyle; Sagi, Veena; Markwarth, Nathan; Chen, Bin; Monteiro, Antónia
Fonte: University of Wisconsin Library Publicador: University of Wisconsin Library
Tipo: Artigo de Revista Científica
Publicado em 25/10/2007 EN
Relevância na Pesquisa
47.34%
The direct transfer of genes into differentiated insect tissues is a useful method of determining gene function because it circumvents the need to perform germ line transformations and of having information on tissue-specific gene promoters. Here in vivo gene delivery is achieved through electroporation of a reporter gene into the pupal forewing of the butterfly Bicyclus anynana (Butler) (Lepidoptera: Nymphalidae). Plasmids containing the coding sequence for enhanced green fluorescent protein (EGFP), driven by the Drosophila heat-shock promoter hsp70, were successfully expressed in epidermal cells after plasmid injection followed by electroporation and heat shock. EGFP expression was restricted to the vicinity of the injection and electroporation site, but the number of transformed cells varied from a few to over 5000 cells. Electroporation parameters were optimized in order to maximize the number of transformed cells while minimizing the extent of damage to the adult wing. While certain electrical parameters were well tolerated by the wing tissue, the physical damage caused by the insertion of the tungsten electrodes led to frequent disruptions of the adult wing pattern around the puncture sites. While this technique can be useful to test the correct expression of marker genes (such as EGFP) in newly build plasmids immediately following their injection...

Male Courtship Rate Plasticity in the Butterfly Bicyclus anynana Is Controlled by Temperature Experienced during the Pupal and Adult Stages

Bear, Ashley; Monteiro, Antónia
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 22/05/2013 EN
Relevância na Pesquisa
47.34%
Environmental cues can act to initiate alternative developmental trajectories that result in different adult phenotypes, including behavioral phenotypes. The developmental period when an organism is sensitive to the cue is often described as a critical period. Here we investigated the critical period for temperature-sensitive courtship rate plasticity in the butterfly Bicyclus anynana. We performed a series of temperature-shift experiments in which larvae, pupae, or adults were shifted for blocks of time from one temperature to an alternative temperature, and then we quantified the courtship rate exhibited by adult males. We discovered that the critical period begins during pupal development and extends into adulthood, but temperature experienced during larval development does not affect male courtship rate. This finding allows us to develop hypotheses that address how developmental and physiological factors may have influenced the evolution of behavioral plasticity in this species.

Sex pheromone biosynthetic pathways are conserved between moths and the butterfly Bicyclus anynana

Liénard, Marjorie A; Wang, Hong-Lei; Lassance, Jean-Marc; Löfstedt, Christer
Fonte: Nature Pub. Group Publicador: Nature Pub. Group
Tipo: Artigo de Revista Científica
Publicado em 27/05/2014 EN
Relevância na Pesquisa
47.34%
Although phylogenetically nested within the moths, butterflies have diverged extensively in a number of life history traits. Whereas moths rely greatly on chemical signals, visual advertisement is the hallmark of mate finding in butterflies. In the context of courtship, however, male chemical signals are widespread in both groups although they likely have multiple evolutionary origins. Here, we report that in males of the butterfly Bicyclus anynana, courtship scents are produced de novo via biosynthetic pathways shared with females of many moth species. We show that two of the pheromone components that play a major role in mate choice, namely the (Z)-9-tetradecenol and hexadecanal, are produced through the activity of a fatty acyl Δ11-desaturase and two specialized alcohol-forming fatty acyl reductases. Our study provides the first evidence of conservation and sharing of ancestral genetic modules for the production of FA-derived pheromones over a long evolutionary timeframe thereby reconciling mate communication in moths and butterflies.

Selection and Validation of Reference Genes for qRT-PCR Expression Analysis of Candidate Genes Involved in Olfactory Communication in the Butterfly Bicyclus anynana

Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 20/03/2015 EN
Relevância na Pesquisa
47.62%
Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods...

Natural Loss of eyeless/Pax6 Expression in Eyes of Bicyclus anynana Adult Butterflies Likely Leads to Exponential Decrease of Eye Fluorescence in Transgenics

Das Gupta, Mainak; Chan, Sam Kok Sim; Monteiro, Antónia
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 14/07/2015 EN
Relevância na Pesquisa
47.34%
Commonly used visible markers for transgenesis use fluorescent proteins expressed at the surface of the body, such as in eyes. One commonly used marker is the 3xP3-EGFP cassette containing synthetic binding sites for the eyeless/Pax6 conserved transcription factor. This marker cassette leads to fluorescent eyes in a variety of animals tested so far. Here we show that upon reaching adulthood, transgenic Bicyclus anynana butterflies containing this marker cassette exponentially loose fluorescence in their eyes. After 12 days, transgenic individuals are no longer distinguishable from wild type individuals. The decreased eye fluorescence is likely due to significantly decreased or halted eyeless/Pax6 expression observed in wild type animals upon adult emergence. Implications from these findings include care in screening transgenic animals before these reach adulthood, or shortly thereafter, and in using adult animals of the same age for quantitative screening of likely homozygote and heterozygote individuals.

Sex pheromone biosynthetic pathways are conserved between moths and the butterfly Bicyclus anynana

Liénard, Marjorie A; Wang, Hong-Lei; Lassance, Jean-Marc; Löfstedt, Christer
Fonte: Nature Pub. Group Publicador: Nature Pub. Group
Tipo: Artigo de Revista Científica
EN_US
Relevância na Pesquisa
47.34%
Although phylogenetically nested within the moths, butterflies have diverged extensively in a number of life history traits. Whereas moths rely greatly on chemical signals, visual advertisement is the hallmark of mate finding in butterflies. In the context of courtship, however, male chemical signals are widespread in both groups although they likely have multiple evolutionary origins. Here, we report that in males of the butterfly Bicyclus anynana, courtship scents are produced de novo via biosynthetic pathways shared with females of many moth species. We show that two of the pheromone components that play a major role in mate choice, namely the (Z)-9-tetradecenol and hexadecanal, are produced through the activity of a fatty acyl Δ11-desaturase and two specialized alcohol-forming fatty acyl reductases. Our study provides the first evidence of conservation and sharing of ancestral genetic modules for the production of FA-derived pheromones over a long evolutionary timeframe thereby reconciling mate communication in moths and butterflies.

A Gene-Based Linkage Map for Bicyclus anynana Butterflies Allows for a Comprehensive Analysis of Synteny with the Lepidopteran Reference Genome

Beldade, P.; Saenko, SV.; Pul, N.; Long, AD.
Fonte: PLOS Publicador: PLOS
Tipo: Artigo de Revista Científica
Publicado em /02/2009
Relevância na Pesquisa
47.34%
Lepidopterans (butterflies and moths) are a rich and diverse order of insects, which, despite their economic impact and unusual biological properties, are relatively underrepresented in terms of genomic resources.We focus on Bicyclus anynana butterflies, a laboratory system for studying the diversification of novelties and serially repeated traits. With a panel of 12 small families and a biphasic mapping approach, we first assigned 508 expressed genes to segregation groups and then ordered 297 of them within individual linkage groups. We also coarsely mapped seven color pattern loci.

Quantitative analysis of Bicyclus anynana's eyespot wing pattern images

Lopes, Pedro dos Santos, 1982-
Fonte: Universidade de Lisboa Publicador: Universidade de Lisboa
Tipo: Dissertação de Mestrado
Publicado em //2011 ENG
Relevância na Pesquisa
58.08%
Tese de mestrado. Bioinformática e Biologia Computacional. Universidade de Lisboa, Faculdade de Ciências, 2011; The main objective of this work is to provide researchers with a tool to quantitatively analyse images of the eyespot patterns present in the wings of the butterfly species Bicyclus anynana. More specifically, this tool is a plugin for imageJ, a free open-sourced image processing program. Until now, researchers have been using software such as imageJ to quantify some dimensions of these patterns through manual measurements. This plugin offers an effective, quick and automatic way to obtain these measurements and others more difficult to get until now, such as the area of each coloured region. It also offers the possibility to obtain representative images of the eyespot(s). Besides images with a single eyespot, the program also analyses wing images with several eyespots as well as fluorescence microscopy images with specific proteins labelled. In the first two types, the program finds the eyespots automatically and analyses them individually, and in the latter ones, the program can provide us with intensity plots of transversal cuts through the middle of the eyespot. To obtain the required data, the plugin finds the centre of each eyes pot and...

Mitochondrial DNA Signature for Range-Wide Populations of Bicyclus anynana Suggests a Rapid Expansion from Recent Refugia

de Jong, Maaike A.; Wahlberg, Niklas; van Eijk, Marleen; Brakefield, Paul M.; Zwaan, Bas J.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 22/06/2011 EN
Relevância na Pesquisa
47.62%
This study investigates the genetic diversity, population structure and demographic history of the afrotropical butterfly Bicyclus anynana using mitochondrial DNA (mtDNA). Samples from six wild populations covering most of the species range from Uganda to South Africa were compared for the cytochrome c oxidase subunit gene (COI). Molecular diversity indices show overall high mtDNA diversity for the populations, but low nucleotide divergence between haplotypes. Our results indicate relatively little geographic population structure among the southern populations, especially given the extensive distributional range and an expectation of limited gene flow between populations. We implemented neutrality tests to assess signatures of recent historical demographic events. Tajima's D test and Fu's FS test both suggested recent population growth for the populations. The results were only significant for the southernmost populations when applying Tajima's D, but Fu's FS indicated significant deviations from neutrality for all populations except the one closest to the equator. Based on our own findings and those from pollen and vegetation studies, we hypothesize that the species range of B. anynana was reduced to equatorial refugia during the last glacial period...

Temporal Gene Expression Variation Associated with Eyespot Size Plasticity in Bicyclus anynana

Oliver, Jeffrey C.; Ramos, Diane; Prudic, Kathleen L.; Monteiro, Antónia
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 10/06/2013 EN
Relevância na Pesquisa
47.34%
Seasonal polyphenism demonstrates an organism's ability to respond to predictable environmental variation with alternative phenotypes, each presumably better suited to its respective environment. However, the molecular mechanisms linking environmental variation to alternative phenotypes via shifts in development remain relatively unknown. Here we investigate temporal gene expression variation in the seasonally polyphenic butterfly Bicyclus anynana. This species shows drastic changes in eyespot size depending on the temperature experienced during larval development. The wet season form (larvae reared over 24°C) has large ventral wing eyespots while the dry season form (larvae reared under 19°C) has much smaller eyespots. We compared the expression of three proteins, Notch, Engrailed, and Distal-less, in the future eyespot centers of the two forms to determine if eyespot size variation is associated with heterochronic shifts in the onset of their expression. For two of these proteins, Notch and Engrailed, expression in eyespot centers occurred earlier in dry season than in wet season larvae, while Distal-less showed no temporal difference between the two forms. These results suggest that differences between dry and wet season adult wings could be due to a delay in the onset of expression of these eyespot-associated genes. Early in eyespot development...

Female Bicyclus anynana butterflies choose males on the basis of their dorsal UV-reflective eyespot pupils

Robertson, Kendra A; Monteiro, Antónia
Fonte: The Royal Society Publicador: The Royal Society
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
47.34%
Sexual and natural selection pressures are thought to shape the characteristic wing patterns of butterfly species. Here we test whether sexual selection by female choice plays a role in the maintenance of the male wing pattern in the butterfly Bicyclus anynana. We perform one of the most extensive series of wing pattern manipulations in butterflies, dissecting every component of the ‘bulls-eye’ eyespot patterns in both ventral and dorsal wing surfaces of males to test the trait's appeal to females. We conclude that females select males on the basis of the size and brightness of the dorsal eyespot's ultraviolet reflecting pupils. Pupil absence is strongly selected against, as are artificially enlarged pupils. Small to intermediate (normal sized) pupils seem to function equally well. This work contradicts earlier experiments that suggest that the size of dorsal eyespots plays a role in female choice and explains why male dorsal eyespots are very variable in size and often have indistinct rings of coloration, as the only feature under selection by females seems to be the central white pupil. We propose that sexual selection by female choice, rather than predator avoidance, may have been an important selective factor in the early stages of eyespot evolution in ancestral Lepidopteran lineages.

Microsatellite markers associated with genes expressed in developing wings of Bicyclus anynana butterflies

Beldade, P.; Van´T Hof, Arje`n E.; Jerónimo, MA.; Long, AD.
Fonte: Blackwell Publicador: Blackwell
Tipo: Artigo de Revista Científica
Publicado em /02/2009
Relevância na Pesquisa
67.68%
Deriving useful microsatellite markers in lepidopterans has been challenging when relying on scans of genomic DNA libraries, presumably due to repetitiveness in their genomes. We assayed 96 of 320 microsatellites identified in silico from a collection of Bicyclus anynana ESTs, in 11 independent individuals from a laboratory population.From the 68 successful assays, we identified 40 polymorphic markers including 22 with BLAST-based annotation. Nine of 12 selected polymorphic markers tested in a panel of 24 wild-caught individuals converted to successful assays and were all polymorphic. We discuss how microsatellite discovery in ESTs is an efficient strategy with important attendant advantages.