Genomic Markers for the Study of Ostrinia Nubilalis Population

Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations

2005 Genomic markers for the study of Ostrinia nubilalis population dynamics and traits conferring resistance to Bacillus thuringiensis (Bt) crystalline toxins Brad Steven Coates Iowa State University

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Recommended Citation Coates, Brad Steven, "Genomic markers for the study of Ostrinia nubilalis population dynamics and traits conferring resistance to Bacillus thuringiensis (Bt) crystalline toxins " (2005). Retrospective Theses and Dissertations. 1855. https://lib.dr.iastate.edu/rtd/1855

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conferring resistance to Bacillus thuringiensis (Bt) crystalline toxins

Brad Steven Coates

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Genetics

Program of Study Committee: Daniel F. Voytas, Co-major Professor Leslie C. Lewis, Co-major Professor Richard L. Hellmich Fredric J. Janzen John D. Nason Jonathan F. Wendel

Iowa State University Ames, Iowa 2005

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TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES vi

ABSTRACT vii

CHAPTER 1. GENERAL INTRODUCTION 1 Introduction 1 Dissertation Organization 2 Literature Review 3 References 31

CHAPTER 2. PARTIAL MITOCHONDRIAL GENOME SEQUENCES OF OSTRINIA NUBILALIS AND OSTRINIA FURNACALIS Abstract 48 Introduction 48 Materials and Methods 49 Results and Discussion 51 References 55

CHAPTER 3. GEOGRAPHIC AND VOLTINISM DIFFERENTIATION AMONG NORTH AMERICAN OSTRINIA NUBILALIS (EUROPEAN CORN BORER) MITOCHONDRIAL CYTOCHROME C OXIDASE HAPLOTYPES Abstract 60 Introduction 60 Materials and Methods 62 Results and Discussion 66 References 73

CHAPTER 4. POLYMORPHIC CA/GT AND GA/CT MICROSATELLITE LOCI FOR OSTRINIA NUBILALIS (LEPIDOPTERA:CRAMBIDAE) Abstract 83 Short Note 83 References 87

CHAPTER 5. TWO SEX-CHROMOSOME-LINKED MICROSATELLITE LOCI SHOW GEOGRAPHIC VARIANCE AMONG NORTH AMERICAN

Abstract 89 Introduction 90 Materials and Methods 91 IV

Results 94 Discussion 95 References 99

CHAPTER 6. SEQUENCE VARIATION IN THE CADHERIN GENE OF OSTRINIA NUBILALIS'. A TOOL FOR FIELD MONITORING Abstract 105 Introduction 105 Materials and Methods 108 Results and Discussion 112 References 119

CHAPTER 7. TRYPSIN- AND CHYMOTRYPSIN-LIKE CDNAS FROM THE MIDGUT OF OSTRINIA NUBILALIS Abstract 135 Introduction 136 Experimental Procedures 138 Results and Discussion 142 References 151

CHAPTER 8. A (3-1,3-GALACTOSYLTRANSFERASE AND BRAINIAC/ BRE-5 HOMOLOG FROM OSTRINIA NUBILALIS Abstract 166 Introduction 166 Materials and Methods 169 Results and Discussion 175 References 180

CHAPTER 9. CONCLUSION 189

ACKNOWLEDGMENTS 198 V

LIST OF TABLES

Table 2.1. Mitochondrial codon usage table. 59

Table 2.2. Nucleotide frequencies in mitochondrial genomes. 59

Table 3.1. Twenty-six variable nucleotide positions observed between 79 14 North American O. nubilalis mitochondrial coxl and cox2 sequences.

Table 3.2. Distribution of mitochondrial RFLP frequencies and 80 haplotypes in samples from 15 North American collection sites.

Table 3.3. Mitochondrial AMOVA and modified ^-statistics. 81

Table 3.4. Significantly different pairwise Nei genetic distance (D) comparison 82 among mitochondrial haplotypes.

Table 4.1. Ten Ostrinia nubilalis CA/GT and GA/CT microsatellite markers. 88

Table 5.1. North American O. nubilalis male ONZ1 genotypic, and heterogametic 103 female ONW1 and ONZ1 haplotype frequencies.

Table 5.2. Male AMOVA and FsT, and female AMOVA and Ost values. 104

Table 6.1. Mendelian inheritance of cadherin alleles in O. nubilalis pedigrees 133 Ped31 and Ped62.

Table 6.2. Mead, Nebraska O. nubilalis frequency of cadherin PCR-RFLP alleles. 134

Table 7.1. Ostrinia nubilalis trypsin- and chymotrypsin-like serine protease 162 gene primers.

Table 7.2. Ostrinia nubilalis midgut cDNA clones and putative derived 163 mature serine protease properties.

Table 7.3. Pair-wise similarity matrix of O. nubilalis midgut expressed 164 serine protease cDNA sequences.

Table 7.4. Test of Mendelian inheritance of trypsin and chymotrypsin alleles 165 in O. nubilalis pedigrees PedlOb and Ped24a. VI

LIST OF FIGURES

Figure 2.1. Ostrinia mitochondrial genome map of sequenced regions. 58

Figure 2.2. Predicted tRNAARG secondary structures. 58

Figure 3.1. Location of 15 North American O. nubilalis collection sites. 77

Figure 3.2. Parsimony haplotype tree constructed from a 2,156 bp 78 mitochondrial DNA alignment.

Figure 5.1. Sex linked microsatellite sequences. 102

Figure 6.1. Ostrinia nubilalis 1717 residue long cadherin-like peptide sequence. 125

Figure 6.2. Lepidopteran cadherin phytogeny. 127

Figure 6.3. Variation in lepidopteran cadherin toxin binding regions (TBRl and 2). 128

Figure 6.4. Alignment of O. nubilalis cadherin CrylA toxin binding region 1 129 (OnTBRl).

Figure 6.5. Alignment of O. nubilalis cadherin CrylA toxin binding region 2 131 (OnTBR2).

Figure 7.1. Trypsinogen and chymotrypsinogen-like protease sequences. 156

Figure 7.2. Lepidopteran trypsinogen and chymotrypsinogen peptide phytogeny. 159

Figure 7.3. Trypsin-(OnT25, OnT23, and OnT3) and chymotrypsin-like 161 (OnCl and OnC2) transcript reverse transcriptase (RT)-PCR detection among tissues.

Figure 8.1. Peptide sequence alignment of invertebrate 185 (5-1,3-galactosyltransferases.

Figure 8.2. A consensus (3-l,3-galactosyltransferase-5/brainiac/BRE-5 phytogeny. 186

Figure 8.3. Relative abundance O. nubilalis (3-1,3-galactosyltransferase 187 (Onb3GalT5) transcripts among larval tissues.

Figure 8.4. Ostrinia nubilalis P-1,3-galactosyltransferase (Onb3GalT5) 188 5'-untranslated and promoter region sequence. vii

ABSTRACT

A candidate gene approach was taken to characterize Bacillus thuringiensis (Bt) crystalline {Cry) toxin resistance phenotypes of the European com borer, Ostrinia nubilalis

(Hiibner), and anonymous genomic markers were used to estimate subpopulation

differentiation and migration rates that may affect spread of resistance phenotypes in the

North American population. Native and transgenic variants of Bacillus thuringiensis (Bt) crystalline {Cry) toxins control larval lepidopteran feeding upon important agronomic crop

plants. Invertebrate Bt resistance phenotypes arise via altered expression of midgut proteases involved in Cry toxin activation (trypsins) and degradation (chymotrypsins), reduced toxin binding to N-acetylgalactoseamine (GalNAc) modified peptide receptors (cadherin, aminopeptidase, or alkaline phosphatase), or receptor glycosylation pathway knockouts (P-

1,3-galactosyltransferases; p3GalT5). Ostrinia nubilalis trypsin, chymotrypsin, cadherin,

and p3GalT5 cDNA clones, transcript expression, and partial genomic DNA copies were

characterized. Mendelian inheritance of trypsin, chymotrypsin, and cadherin genomic DNA

markers suggest association between allele segregation and Bt resistance phenotypes can be

conducted. Movement of O. nubilalis moths was indirectly estimated by genetic similarity

between North American subpopulations using mitochondrial and genomic microsatellite

markers. Marker data suggested differentiation between two O. nubilalis ecotypes,

univoltine and bivoltine, which may affect movement of Bt resistance phenotypes in northern regions of the Midwest United States. 1

CHAPTER 1. GENERAL INTRODUCTION

1.1 Introduction

Insect pests of cultivated crops cause economically detrimental damage to plant and yield worldwide. The European com borer, Ostrinia nubilalis, larvae are a major insect pest of com in Europe and North America. Introduction of transgenic maize expressing versions of the Bacillus thuringiensis (Bt) CrylAb toxin gene as a crop production option has raised concern for resistance development in target insects, similar to what historically has occurred with chemical pesticides.

Evaluation of potential widespread resistance to transgenic com hybrids requires 1) knowledge of genetic structure of O. nubilalis in North America, and 2) identification of genes involved in the resistance mechanism. Selectively neutral genomic markers are necessary for accurate estimation of moth movement (migration), inbreeding, and population substructure. Hypothetically, high migration levels and lack of population substructure should facilitate rapid spread of resistance phenotypes, whereas low migration rates and a highly structured population may lead to pockets of resistance that will not spread rapidly to the population as a whole.

Identification of biological candidate genes that may function in the O. nubilalis Bt resistance mechanism, and subsequent development of gene specific molecular markers, may allow evaluation of genotypic association with resistance phenotypes. High correlation between alleles of a candidate Bt resistance gene and high larval tolerance of Bt toxin may suggest gene involvement in the phenotype. Tracking of resistance alleles in the O. nubilalis population, in conjunction with population studies, can be to evaluate mechanism and dissemination rate of Bt resistance phenotypes. 2

1.2 Dissertation Organization

This Dissertation is organized into eight chapters. Chapter 1 presents background information on genome organization and population biology and genetics of O. nubilalis.

Additionally, Chapter 1 addresses prior studies of B. thuringiensis resistance mechanisms in

Lepidoptera, nematodes, and O. nubilalis.

The remaining seven chapters outline studies performed in my graduate work to identify molecular genetic markers for the study of O. nubilalis population dynamics and characterization of candidate B. thuringiensis resistance genes in the North American population. Chapter 2 presents the DNA sequencing of the mitochondrial DNA from O. nubilalis and its sister species O.furnacalis, from which regions of high variability were identified. Subsequent mitochondrial RFLP markers for the differentiation of O. nubilalis haplotypes are shown in Chapter 3, with specific comparison of two different ecotypes.

Chapter 4 and 5 describes the variation of 12 O. nubilalis microsatellite markers. Two sex- linked microsatellites in Chapter 4, one each on the Z- and W-chromosomes, suggest differentiation between sample sites from South Dakota and Minnesota compared to those from Iowa and Minnesota. A short research summary presenting the levels of genetic variation among 10 CA/GT and GA/CT microsatellite makers from a Crawfordsville, Iowa population is Chapter 5.

Chapters six through eight involve isolation of candidate Bt resistance genes, cadherin, trypsins and chymotrypsins, and a (3-1,3-galactosyltransferase (|33GalT5) bre5/brn from O. nubilalis. Cadherin cDNA isolation of development of genomic DNA markers for phenotypic association mapping is addressed in chapter 6. Identification of three trypsin-like and two chymotrypsin-like genes expressed in the midgut, and Mendelian inherited genomic 3 markers for each gene are described in chapter 7. Finally, the cDNA and genomic sequence for a (3-1,3 galactosyltransferase, homologous to Bt resistance gene 5 (bre5) in

Caenorhabditis elegans and brainiac (brn) in Drosophila melanogaster, is shown in chapter

8 along with preliminary data on variation of genomic markers.

1.3 Literature Review

1.3.1 Taxonomy and Phylogeny

Ostrinia nubilalis, the European com borer (ECB), is a lepidopteran insect species from the superfamily Pyralinae and family Crambidae (Fletcher and Nye 1984; Common

1990; Solis and Mitter 1992). Pyralids are a collection of ill-defined moths that are small in size, and commonly referred to as snout or grass moths (Borror, Triplehom, and Johnson

1989). The superfamily includes over 30,000 species (Monroe 1972) with the synapomorphic character of paired tympanum located on the first abdominal segment of the adults (Minet 1982) and triangular forewings (Borror, Triplehom, and Johnson 1989).

Mutuura and Monroe (1970) classified three groups with the genus Ostrinia based on differences in male genitalia. Ostrinia nubilalis males have a trilobed uncus, which is a character shared with O. scapulalis, O. zaguliaevi, O. orientalis, and O.furnacalis. Fore tibial size separates O. nubilalis, O. scapulalis, O. orientalis, and O.furnacalis from the remainder, but no definitive external morphological features are present to allow further classification.

A 682 bp DNA sequence alignment from the mitochondrial cytochrome c oxidase subunit II gene (COII) showed 0.15 to 2.38% sequence difference and provided suitable characters for phylogenetic analysis (Kim et al. 1999). Interspecific comparison showed 71 4 variable nucleotide sites; with 60% of those being transitions and 68% were at third codon positions. Sequences were 76 to 77.4% A or T nucleotides. A neighbor-joining (NJ) tree was construction using the Jukes-Cantor (JC; 1969) model of sequence evolution indicated, and its topology conflicted with cladiograms constructed from morphological data. The NJ tree by Kim et al. (1999) failed to place all large tibia species, O. nubilalis, O. scapulalis, O. orientalis, and O.furnacalis, into the same clade. Reason for incongruence might lie in incorrect phylogenetic modeling, since the JC model assume 1:1:1:1 ratio of nucleotides, and equal likelihood of transition or transverion mutation. The aforementioned results by Kim et al. (1999) indicate a Kimura 2-paramter (K2P) model was more appropriate (transition to transversion ratio of 2:3 and 76% AT composition).

1.3.2 North American Introductions and Intraspecific Polymorphism.

Species Introduction to North America

Ostrinia nubilalis larvae are polyphagous lepidopteran pests of cultivated maize in

North America (Showers 1993). The species was first introduced to North America from

Europe in the early 1900's and collected from sweet com near Boston, MA (Vinal 1917; Felt

1919), and was hypothesized to have arrived on shipments of Hungarian or Italian broomcom

(Smith 1920). Vinal and Caffrey (1919) described the Massachusetts population as having two generations per year. Multiple separate introductions, migrations, or transports likely occurred along the east coast of the United States and southeastern Canada in subsequent years (Poos 1927; Showers 1993), with a Welland, Ontario introduction coming from infected com (Spencer and Crawford 1923). Infestations were observed in the region 5 surrounding Lake Erie between 1919 and 1921 (Caffrey and Worthley 1927), but these

populations showed only a single reproducing generation per year that supported the idea that

a unique introduction of genetically distinct O. nubilalis had occurred (Brindley and Dicke

1963).

Migration south and west occurred gradually from 1927 to 1936. Rapid spread took place in the following years, at a westward rate of approximately 114 km per year (Palmer et al. 1985). Moths were observed in the prairie provinces of Canada by 1949, North Dakota by

1950 (Chiang 1972), and the Rocky Mountain and Gulf States by the early 1950s. This

expanding population mostly was comprised of bivoltine moths (Vance 1942; Bird and

Stewart 1959). Movement of univoltine moths to northern regions might have lagged that of

bivoltine moths. Chiang (1972) showed since an initial O. nubilalis infestation in Minnesota

and South Dakota by 1943, two distinct waves of secondary invasions occurred in 1952

(univoltine) and 1966 (bivoltine). Contemporary dispersal patterns of voltinism ecotypes are determined by genetic and environmental factors that establish north-south clines.

Larval O. nubilalis initially were described as infesting many host plants (Vinal and

Caffrey 1919). In its native range O. nubilalis fed principally upon hop (Humulus lupulus

L.), sagebrush (Artemisia sp), and mugwart (A. vulgaris; Caffrey and Worthley 1927;

Thompson and Parker 1928). Corn is the preferred host plant for O. nubilalis. Potato plants also are attractive for first generation oviposition, whereas sorghum, cotton, and vegetable crops are targets of second-generation corn borers. Economically detrimental levels of crop damage occurred to com, with a peak between 1925 and 1927 (Brindley and Dicke 1963).

Weed species have capacity to sustain larval development (Hodgson 1928; Dicke 1932). 6

Intraspecific Polymorphism in Pheromone Production and Reception

Ostrinia nubilalis, the European corn borer, is sexually dimorphic with the males being smaller, darker, and having pointed abdomens, whereas females are larger, cream colored, and have large rounded abdomens. Female pheromone glands produce isomeric forms (E- and Z-) of Al 1 -tetradecenyl acetates (£11- and Zll-14:OAc; Klun et al. 1975).

Pheromone blends show intraspecific polymorphism with respect to major component, with

E-strain (New York type) females emitting an E\Z blend of 99:1, and Z-strain (Iowa type) produces a 3:97 ratio (Klun et al. 1973; Kochansky et al. 1975) or 98:2 (Glover et al. 1987).

Genes associated with male pheromone response are located on the Z chromosome and linked to the allozyme marker triose phosphate isomerase (TPI; Glover et al. 1990). An autosomal locus determines male pheromone receptor sensilla organization and affects male sex pheromone response (Roelofs et al. 1987). Two different sensilla on male antenna are characterized as transmitting with large or small spike amplitude, with Z pheromone strain high amplitude sensilla "tuned" to the Z isomer and visa versa. Ratio of stereoisomer^ pheromone by females is controlled by a single autosomal locus (Roelofs et al. 1987), and mapped to linkage group 12 (Dopman et al. 2004). Autosomal loci controlling male sensilla arrangement and female pheromone production were not mapped by Dopman et al. (2004), but shown to be outside the same linkage group (Lôfstedt et al. 1989).

The Z-pheromone-emitting and responding populations are distributed in the central and eastern United States and southern Canada (Showers et al. 1974), whereas the ^-strain is in the northeastern United States and Quebec (McLeod et al. 1979). Distribution of pheromone strains overlap in the eastern United States where intermating of pheromone races was documented by isolating female blend intermediates from field populations (Roelofs et 7

al. 1985; Durant et al. 1995), which were similar to blends produced by F2 hybrids in laboratory crosses (Roelofs et al. 1985).

Intraspecific Polymorphism in Diapause Induction

Voltinism and diapause: Diapause is defined as the state of retarded development that is determined by genetic composition and induced by the environment. The 5th instar O. nubilalis bore into stalks of host plants in late summer to early autumn in preparation for a hibernation-like diapause state. Over winter survival in diapause is associated with physiological changes; increases of glycerol, and storage and transport proteins in the hemolymph (Palli et al. 1993). Larvae in diapause appear bloated and lethargic. Beck and

Hanec (1960) further identified three diapause characteristics, 1) arrest in development of the gonads, 2) reduction in respiration, and 3) failure to pupate after late instar feeding stopped.

The diapause state also is characterized by brain inactivity, and non-response of the prothoracic glands to PTTH activation (Richards and Saunders 1986; Gelman et al. 1992).

Juvenile hormone (JH) may play a major role in diapause maintenance, and metamorphosis

(Yin and Chippendale 1976), and suppression of 20-hydroxyecdysone is crucial in delay of molting during diapause (Denlinger 1985). Proctodone, a photoperiod sensitive developmental hormone secreted by epithelial cells of the com borer ilium, was hypothesized to have a role in diapause induction (Beck and Alexander 1964; Beck et al. 1965).

Voltinism ecotypes of O. nubilalis show differing larval response to environmental cues (photoperiod and temperature) for induction of diapause (Showers 1993; Mason et al.

1996). Showers et al. (1975) determined that northern (univoltine), central (bivoltine), and southern (multivoltine) ecotypes exist based upon diapause response. Univoltine ecotypes 8 have one diapause cycle per calendar year, bivoltine two diapause periods, and multivoltine with more than two. Arbuthnot (1949) investigated climatic relationship with O. nubilalis generation number, and showed that a CT population was a pure multivoltine strain, whereas an OH sample was mixed uni- and bivoltine. Mating studies also suggested univoltine-like diapause strain characteristics were genetically recessive. Chiang et al. (1968) showed larvae from Portageville, MS required significantly few degree-days for development and were more responsive to photoperiod compared to those from Waseca, Minnesota. At 23 to 25

°C, the threshold photoperiod to induce diapause was 15.4 h in a 24 h cycle (Beck and Hanec

1960). Diapause was triggered in a Morris, MN population at photophase of 14 h 41 m to 14 h 12 m, and triggered in individuals from an Ankeny, IA population at 14 h 22m to 13 h 55 m

(Showers et al. 1972), showing early onset of diapause by northern univoltine populations.

Also, Fi hybrids from Minnesota and Alabama population crosses showed an intermediate number of degree-days prior to cessation of diapause, and confirmed voltinism traits are male sex (Z-chromosome) linked. McLeod (1976) performed crosses between univoltine individuals from northern with bivoltine moths from southern Ontario, and suggested duration of diapause was, in agreement with Arbuthnot (1949) and Showers et al. (1972), male sex-linked. Showers et al. (1979) found a significant difference in the number of degree days required to break diapause between Concord, MA and Toledo, OH collected larvae. Interestingly the Toledo, OH population recorded a degree-day requirement similar to a bivoltine Muscatine, IA population, suggesting a genetic shift or subsequent invasion of bivoltine moths in the 30 years since the Arbuthnot study.

Data presented above indicate voltinism ecotypes differ in response to environmental cues for entrance into diapause. Genetic differences also are manifested in timing of 9

diapause exit and rate of development into adults. Larvae from multivoltine populations

cease diapause and develop to adults ahead of those from univoltine populations (McLeod

1976). Early emergence of uni- and bivoltine moths is due to a difference in number of

degree-days required post-diapause prior to pupation were observed between voltinism

ecotypes (Showers 1979). This variation in post-diapause developmental time was shown

between voltinism ecotypes and includes both genetic and environmental variance

components (Calvin and Song 1994; Hoard and Weiss 1995). Similar to diapause induction,

post-diapause development traits showed paternal inheritance (Showers et al. 1972; Reed et

al. 1981; Showers 1981).

Genes that favor univoltine ecotypes likely are under positive selection by northern

climatic conditions, which may be an evolutionary adaptation to ensure over-winter survival.

Early onset of winter conditions may not allow sufficient time for second-generation

bivoltine larval development, and climate might be a natural barrier to northern bivoltine

range expansion. The same genes would be culled from southern populations because

extended larval diapause in summer months would make individuals more susceptible to

prédation and parasitiodism (Sparks et al. 1966). Two adaptations, 1) critical photoperiod to

induce diapause (scotophase), and 2) number of degree-days required to break diapause in

the spring were hypothesized to maintain univoltine populations in northern corn growing

regions (McLeod 1976). Univoltine O. nubilalis genotypes are predicted to have selective

advantages in northern climates where short growing seasons favor single generations

(Showers 1993). Resulting north-south dines are observed with univoltine ecotypes residing only in northern ranges, and "barrier latitudes" that inhibit southern migration of univoltine

and northern movement of multivoltine ecotypes were hypothesized (Showers 1979; Showers 10

1993). The movement of bivoltine ecotypes into univoltine regions of MN without reciprocal migration supported the barrier latitude theory (Chiang et al. 1965, McEwen et al. 1968).

Interesting questions regarding gene flow present themselves in geographic regions where different voltinism ecotypes coexist. Such overlap regions exist in the northern com belt (Showers 1993). In natural conditions, individuals from bi- and multivoltine populations break diapause earlier than univoltine larvae, leading to existence of separate periods of adult flights in some areas of sympatry. Eckenrode et al. (1983) indicated there is overlap in uni- and bivoltine flight times in NY populations. Variation in post-diapause developmental time between voltinism ecotypes and environmental conditions could result in these different adult emergence times (Calvin and Song 1994, Hoard and Weiss 1995), suggesting asynchrony of mating periods in some regions minimizes genetic exchange (gene flow; Roelofs et al. 1985).

Assortative mating due to temporal population substructuring may result, but molecular genetic marker studies are inconclusive (see section 1.1.2). But hybridization between uni-

(Z-pheromone strain) and bivoltine (E-pheromone strain) ecotypes was suggested by isolation of pheromone strain hybrids in geographic regions devoid of bivoltine Z-pheromone strain individuals (Glover et al. 1991).

Westward migration of bivoltine moths in OH occurred at an approximately rate of

114 km per year (Palmer et al. 1985), but univoltine expansion rates were much slower at about 19 km per year. Local adaptation of univoltine phenotypes to day length and temperature at a particular latitude results in selection for timing of diapause induction and duration of winter dormancy. This provides evidence that populations are adapted to environmental conditions of specific geographic ranges, and local adaptation therefore affected migration rate across north-south dines (Showers 1993). Univoltine movement was 11 slower due to one migrating generation per year, sex linkage (females show male voltinism

V traits Z W), and recessive inheritance (1/4 of individuals in the F2 generation will be univoltine and diluted further via inbreeding).

Host Plant Range

Immature stages of O. nubilalis are polyphagous, and feed upon over 100 plants native to Europe or North American (Hodgson 1927). In its native Europe the species fed principally upon hop (Humulus lupulus L.), sagebrush (Artemisia sp), and mugwart (A. vulgaris; Caffrey and Worthley, 1927; Thompson and Parker, 1928). In North America it was reported to infest 223 species of plant (Lewis 1975), and most wild or cultivated plant with stems sufficiently large for larvae to bore (Hudson et al. 1989). Larval O. nubilalis feed upon host plant leaves, bore into stalks prior to pupation, and cause reduced photosynthetic and grain yield capacity. The economic cost of O. nubilalis feeding upon cultivated plants was estimated to be $1 billion per year in the United States (Ostlie et al. 1997).

1.3.3 Population Genetics

Ostrinia nubilalis has 31 pair of autosomes and a single pair of sex chromosomes

(Guthrie et al. 1965; Dopman et al. 2004), where females are heterogametic (ZW) and males are homogametic (ZZ; Traut and Marec 1997). Observed total genome size of Lepidoptera varied from 290 Mb for Danaus plexippus (Lepidoptera Danaidae: the monarch butterfly) to

1940 Mb for Euchlaena irraria (Lepidoptera: Geometridae; Gregory and Herbert 2003), but direct size estimation for O. nubilalis is yet to be made. Lepidoptera have a high number of small chromosomes that comprise their genome, and centromeres are "diffuse" so spindle 12 fibers attach along the entire chromosome (Griffiths et al. 1996). Chromosomes are highly condensed, appear dot-shaped at most meiotic and mitotic metaphase stages, and lack distinguishing features that make individual chromosome identification difficult. The result has been a limited application of cytogenetic tools for genetic and molecular studies.

Molecular genetic markers have been developed for O. nubilalis, and applied to population genetic and genome mapping studies.

Pheromone Strain Differentiation

E- and Z-pheromone strains differentiation was investigated using polymorphic allozyme markers, with low but significant allozyme differentiation was observed (Harrison and Vawter 1977; Carde et al. 1978; Cianchi et al. 1980; Glover et al. 1991). Glutamate oxaloacetae transaminase (GOT-1 and -2), phosphohexose isomerase (PHI), and phosphoglucomutase (PGM) allozyme allele frequencies were significantly different between

E- and Z-pheromone strains collected from Amity Hall, PA (Carde et al. 1978). All allozyme studies suggested partial reproductive isolation with limited interstrain gene flow, but intraspecific gene flow was not investigated. Molecular data supported observations of female blend intermediates in laboratory tests and field populations (Liebherr and Roelofs

1975, Roelofs et al. 1985, Glover et al. 1991; Durant et al. 1995), which indicated hybridization occur. Laboratory choice tests indicated only 6 (c?Z- x ÇE) to 10% (d'E- x

$Z) of confined matings produced interstrain E- to Z-pheromone hybridizations, compared to intrastrain controls (76 to 80%; Carde et al. 1978).

Genes associated with male pheromone response are and linked to an allozyme marker, triose phosphate isomerase (TPI), located on the Z-chromosome Glover et al. 1990). 13

Chiang et al. (1980) found all E-pheromone strain individuals from Geneva, NY were fixed for the Tpi-1 allele, which agreed with data from six ^-pheromone strain samples from NY

(Glover et al. 1991). Glover et al. (1991) further indicated mating between pheromone strains is unidirectional (c?BE x 2Z-). This was inferred from all 13 F, hybrid females from field populations showing a Tpi-1/-1 genotype, and absence of Tpi-2 alleles from Z- pheromone strains; which is expected under random mating. Willet and Harrison (1999) provided supporting evidence from pheromone binding protein gene comparisons. Since no significant DNA sequence differences of fixed polymorphism was observed between pheromone strains, continued exchange of genes was suggested.

Voltinism Differentiation

Gene flow between uni- and bivoltine ecotypes also was assumed from Tpi marker

(Glover et al. 1991) and trap data from the Atlantic coast of the United States. Bourguet et al. (2000a) sampled 29 French sites and found no differentiation between northern

(univoltine) and southern (bivoltine) populations using six allozyme markers; PGM, TPI, mannose-6-phosphate isomerase (MPI), hydroxybutyrate dehydrogenases (HBDH), glucose-

phosphate isomerase (GPI), and aspartate-aminotransferases (AAT). Estimated 6>(FST; Weir and Cockerham 1984) values from French populations were between 0.003 and 0.034 (mean

= 0.011), and although 6 values were significant observed most of the genetic variation occurred at the within-site level. Therefore, Bourguet et al. (2000a) suggested no real differentiation was present and a panmictic French population existed. Conclusions by

Bourguet et al (2000a) are contrary to the "low but significant" allozyme differentiation 14 between E and Z pheromone strains observed by (Harrison and Vawter (1977), Carde et al.

(1978), and Cianchi et al. (1980).

Microsatellite loci contain tandem repeats of a 2 to 6 nucleotide unit (Chambers and

MacAvoy 2000) that are ubiquitous across genomes, and have been isolated from insect species Drosophila melanogaster (Goldstein and Clark 1995; S chug et al. 1998), Bombyx mori (Damodar et al. 1999), and Apis mellifera (Estoup and Angers 1998). Polymorphic microsatellite alleles arise predominantly by DNA polymerase slippage during lagging strand chromosomal replication (Schlotterer and Tautz 1992) that adds/deletes repeat units

(Stepwise mutation model, SSM; Levinson and Gutmanl987) one at a time (single step mutation model; SSM) or blocks of repeats (multistep mutations; Weber and Wong 1993;

DiRienzo et al 1994). Mutation rates are high; 10"6 to 10"2 per generation have been estimated (Bowcock et al. 1994; Eisen 1999), making microsatellites useful for genetic markers (Litt and Luty 1989; Coates et al. 2001), use in paternity testing (Queller et al.

1993), linkage mapping (Dietrich et al. 1994; Dib et al. 1996), and population genetic studies

(Goldstein and Scholtterer 1999).

Based on two sex-linked microsatellite loci (OnZl and OnWl), females (ZW) collected from northern regions of the central United States (Minnesota and South Dakota) showed significant subpopulation differentiation compared to subpopulations from Indiana,

Iowa, Nebraska, and Kansas (F$T = 0.271, P < 0.0001), and inbreeding within subpopulations

(Fis = 0.241, P < 0.0001). The same data set also suggested female uni- and bivoltine

collections from the Midwest United States were significant (FST - 0.227, P < 0.0001) based on voltinism ecotype (Coates and Hellmich 2003; Chapter 4). Coates and Hellmich also showed genetic differentiation between populations from South Dakota and sympatric 15

Minnesota sites compared to more southern populations, suggesting regional differentiation may be due to presence of homogeneous univoltine populations in South Dakota and intermating of ecotypes at the Lamberton, Minnesota location. Males collected from the same locations, but screened for the OnZl marker only, indicated lower levels of subpopulation differentiation based on identical regional (FST = 0.044, P < 0.0001) and voltinism divisions (FST = 0.028, P < 0.0001).

Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of four mitochondrial fragments showed no polymorphism between O. nubilalis pheromone or voltinism ecotypes from North America (Marcon et al. 1999). Moreover Marcon et al.

(1999) failed to find any polymorphic sites in the nearly 1500 bp of mitochondrial DNA or

511 bp internal transcribed spacer (ITS) investigated. These results are inconclusive, and cannot be used to suggest genetic homogenization of voltinism ectypes. Fifteen variable nucleotide sites were identified in mitochondrial cytochrome c oxidase subunit I (cox I) and

II (cox II) genes, and 4 were present in restriction endonuclease sites (Sau3Al, Sspl, Vspl, and Alul\ Martel et al. 2003). Six mitochondrial haplotypes were identified among 254 individuals collected from France, but one haplotype predominated and was fixed at four of six sample site locations. No comparison of voltinism ecotypes was made, but it suggests a high rate of mitochondrial DNA fixation (Hartl and Clark 1997). Coates et al. (2004;

Chapter 3) identified 9 mitochondrial DNA haplotypes using 4 polymorphic marker loci

(Ddel, Haelll, Mspl, and Sau3AT) located in cox I and II genes, and a single restriction endonuclease resistant haplotype present among 90% of 1414 individuals from 15 United

States sample locations. A Haelll PCR-RFLP marker was not present among samples from the Atlantic coast, whereas 5.1% of midwestem United States individuals showed the 16 haplotype, and low but significant haplotype frequency differences were detected between sympatic uni- and bivoltine Z-pheromone strains from two MN collection sites (FST = 0.030,

P = 0.008). Significant pairwise genetic distance estimates also were observed between sympatic ecotypes from Lamberton, MN (D = 0.0534, P < 0.0001) using a Bonferroni corrected threshold of 0.0003.

PCR-random amplification of polymorphic DNA (RAPD) analysis by Pomkulwat et al. (1998) indicated genetic separation of a multivoltine subpopulation (TN) from a univoltine (SD) and a bivoltine ecotype subpopulation (MI), suggesting differentiation and barriers to gene flow between voltinism ecotypes. No significant variance in fragment composition existed between uni- and bivoltine ecotypes, but differences reported by

Pomkulwat et al. (1998) may be geographic- instead of ecotype-based.

To date, field data suggest overlap of uni- and bivoltine adult flight times (Eckenrode et al. 1983), genetic homogenization (Bourguet et al. 2000a), and hybridization (Glover et al.

1991), or significant separation of adult emergence and genetic differentiation (Coates et al.

2003, Chapter 5; Coates et al. 2004b, Chapter 3). Sympatic differentiation among O. nubilalis based on host plant preference was shown between larvae developing on sagebrush

(Artemisia sp) and mugwart (A. vulgaris) and those on Zea mays (Bourguet et al. 2000b,

Martel et al. 2003).

Two O. nubilalis voltinism ecotypes in North America and may constitute a barrier to gene flow within the species, and be a most pertinent factor affecting movement of CrylAb toxin resistance alleles. Voltinism subdivision occurs in major maize production areas in the

Midwest U.S. states of Iowa, Illinois, Minnesota, Missouri, Nebraska, South Dakota, and

Wisconsin. Regions of Minnesota, South Dakota, and Wisconsin have sympatic 17 subpopulations that will be key for analysis of intereeotype genetic exchange in absence of confounding geographic effects. Secondly, sympatic voltinism ecotypes regions of

Minnesota and South Dakota co-occur with areas of high percentages of transgenic maize acreages. The co-occurrence of temporally separated sympatic subpopulations and high selection pressure suggests potential mating isolation may provide a barrier to flow of alleles conferring resistance to CrylAb toxins. Genetic barriers between voltinism ecotypes

(temporal population structure), recent imposition of strong selection pressure for survival on

Cryl Ab-expressing transgenic maize, and capacity for rapid adaptation by O. nubilalis makes it an ideal candidate for study of ecotype differentiation and migration-selection balance.

Variation Based on Host Plant

Ostrinia nubilalis is a polyphagous lepidopteran pest of cultivated maize in North

America (Mason et al. 1996). In its native Europe the species fed principally upon hop

(Humulus lupulus L.), sagebrush (Artemisia sp), and mugwart (A. vulgaris; Caffrey and

Worthley, 1927; Thompson and Parker, 1928). In North America was reported to infest 223 species of plant (Lewis 1975), and most wild or cultivated plant with stems sufficiently large for larvae to bore (Hudson et al. 1989).

Ecotypes of O. nubilalis were shown to differentially infest different cultivated crop plants. The bivoltine E-pheromone strain damaged apple shoots and snap bean crops in New

York with a greater frequency than did uni- or bivoltine larvae of the Z-pheromone strain

(Straub et al. 1986; Eckenrode and Webb 1989). Bourguet et al. (2000b) found larvae collected from maize showed significant genetic differentiation with larvae collected on 18

sagebrush (0= FST = 0.057, P < 0.0001) or hop (0= 0.024, P < 0.0001). Larvae collected from hop had greater between hosts (0= 0.024) than within host differentiation (0= 0.002) when compared with maize-derived larvae. Similarly, larvae from sagebrush had greater differentiation compared to maize (0= 0.057) than others collected from sagebrush (0=

0.021). Bourguet et al. (2000b) concluded possible reproductive isolation exists between O. nubilalis populations feeding on maize and those feeding on hop or sagebrush. A follow-up study by Martel et al. (2003) collected mugwart-infecting O. nubilalis from 16 sites, and maize-infesting larvae from 9 sites in France. Allozyme data analysis suggested significant genetic differentiation between plant host groups (0= 0.044). Martel et al. (2003) indicated a significant deficit of heterozygosity at the Mpi locus only for O. nubilalis collected from mugwart, and the highest level of differentiation between plant hosts (0= 0.122). These data suggested host dependent selection was acting upon the Mpi locus or linked loci.

1.3.4 Bacillus thuringiensis (Bt) Toxin Resistance

Ostrinia nubilalis Phenotypic Tolerance to Bt Toxins

Widespread adoption of transgenic CrylAb toxin-expressing plants has heightened concerns of insect resistance development, similar to history of chemical insecticides (Henkel et al. 1997). Scientific and industry leaders devised a "high-dose/refuge strategy" to delay resistance development of insect pests targeted by transgenic com plants (Alstad and Andow

1995). The strategy prescribes; 1) assuming recessive resistance traits, transgenic Bt toxin expression levels need be sufficient to kill 100% of homozygous susceptible (SS) and heterozygous resistant moths (Ss), and 2) larval development and adults emergence from non-Bt com or other appropriate host plant (refuge) should be ensured to mate with 19 homozygous resistant individuals (ss) that develop in transgenic fields. Rare resistant phenotype moths (ss genotype) emerging from transgenic fields should have responsible genes diluted within the population by high probability of mating with susceptible individuals (SS or Ss; Roush 1997; Matten et al. 2004).

Success of the high-dose/refuge strategy depends upon narrow sense heritability (h =

VA/VP), which determines degree to which offspring share phenotypic similarity with parents, or strength of connection between phenotype and genotype. If phenotypic variation

(VP) is highly affected by environmental variance (VE) the influence of additive genetic

2 variance (VA; VG = VD + VA). Realized heritability (h = R/S; R = response to selection, and

S = selection differential between parents and progenitor phenotypes) of Bt toxin resistance in two LA collections and three KS collections of O. nubilalis ranged from 0.17 to 0.31 and

0.36 to 0.46, respectively Huang et al. (1999a). Estimates suggest, at a maximum, 31 to 46% of observed Bt resistance phenotypes are genetically heritable, and the remainder can be partitioned between maternal provisioning and environmental effects.

Resistance of O. nubilalis larvae to Dipel®, a commercial formulation of several Bt toxins, developed quickly in 5 colonies tested by Huang et al. (1997), the trait showed partial dominance (Huang et al. 1999b), and was stable in absence of selection pressure (Huang et al. 1999a). Furthermore resistance levels within the colonies reached a plateau of 40 to 70- fold resistance compared to susceptible isolines after 12 to 20 generations of selection

(Huang et al. 1999a). Susceptibility of larval O. nubilalis to transgenic CrylAb shows geographic and intergenerational variation that was attributed to maternal and environmental influence on resistance traits (Siegfried et al. 1995; Marcon et al. 2000), suggesting a weak connection between parent and offspring phenotypes, and strong influence of environmental 20 factors on expression of resistance traits. Moderate levels of resistance were observed in O. nubilalis laboratory colonies after 3 to 5 generations of recurrent CRYIAb selection for 5 colonies (Huang et al. 1999), and 17 generations or 14 generations of CRY1 Ac selection

(Bolin et al. 1999). Two lines, S-I and S-II, respectively showed 162 and 58-fold increases in

CRYIAc resistance compared to susceptible controls, and susceptibility of S-I decrease to nearly 10-fold may have been due to random genetic drift or negative selection due to inbreeding. Furthermore, Bolin et al. (1999) suggested segregation of alleles at multiple loci could explain intergenerational variation of resistance traits observed in both lines. Onstad and Gould (1998) suggested late season infestation by 5th instar bivoltine occurs at plant senescence CRYIAb titers decrease, and is a threat to the high dose strategy. Estimates of resistant phenotypes in natural populations are low (< 9.2 x 10"4; Andow et al. 1998; Andow et al. 2000; Bourguet et al. 2003).

Insecticide Resistance

Insect resistance to control agents has implications for agricultural production and human health. Pesticide resistances have been acquired by mosquito species Culex tritaeniorhynchus (yellow fever vector; More et al. 2001), Aedes aegypti (yellow fever and dengue fever vector; Yan et al 1998), and Anopheles gambiae (malaria vector; Etang et al.

2003). Agricultural pest species also have developed resistance to field applied control agents. The western com rootworm (Diabrotica sp.; Miota et al. 1998; Zhu et al. 2001), and

Indian mealmoth (Plodia inlerpunctella; McGaughey 1985) and diamondback moth (Plutella xylostela\ Tabashnik et al. 1990) show resistance to carbaryl insecticides and Bacillus thuringiensis (Bt) subsp. kurstaki toxins, respectively. Understanding mechanisms of 21 resistance development and subsequence movement of responsible alleles within populations provides opportunities of understand dynamics of population genetic processes of selection, migration, and evolution of population (ecotype) subdivision.

Resistance has developed in areas of high pesticide use resulting in yield loss in areas harboring tolerant populations (McGaughey and Beeman 1988). Moreover, insecticide resistance has developed by several mechanisms. Nervous system acetylcholinesterase, the target of organophosphorous (OPs) and carbamate insecticides, and sodium channel peptides, the target of organochlorines (DDT) and pyrethroids, show a gain of insect resistance by single amino acid changes (Williamson et al. 1996; Miyazaki et al. 1996). Similarly, cyclodiene resistance occurs by a single nonsynonymous point mutation in the a- aminobutyric acid (GABA) receptor gene (ffrench-Constant et al. 1993). Five point mutations in an acetylcholinesterase insecticide-binding site cause different degrees insecticide resistance (Mutero et al. 1994). Metabolic detoxification of chemical insecticides can occur by action of oxidase, esterase, and glutathione S-transferase (GST) enzyme families. Esterase detoxification is the most common resistance mechanism, which likely is due to large number of gene family members in resistant individual that arises via tandem gene duplication (Vaughan et al. 1997). Oxidases (cytochrome P450 oxidases) also are members of large gene families that manifest themselves into resistant phenotypes via single gene over-expression (Tomita and Scott 1995; Carino et al. 1994).

Lepidopteran Bacillus thuringiensis (Bt) Toxin Resistance Mechanisms

Bacillus thuringiensis (Bt) Berliner is a gram-positive soil bacterium originally described as an insect pathogen against Lepidoptera, Diptera, and Coleoptera (Beegle and 22

Yamamoto 1992). Insoluble inclusion bodies in sporulated B. thuringiensis strains contain

one or more 130 to 140 kDa 8-endotoxin proteins that are plasmid encoded or present on

transposons. Conjugation and recombination between plasmids may contribute to great

endotoxin diversity, and broad range of specificities (Schnepf et al. 1998). The 5-endotoxin is commonly referred to as crystalline (Cry) protein due to crystal structure formed within the insoluble inclusions of Bacillus strains (Hôfte and Whiteley 1989). The native toxin requires proteolytic cleavage into a 60 to 65 kDa active toxin. Active toxins bind receptor molecules along the midgut lumen of susceptible larvae, leading to membrane insertion and subsequent ion channel formation. Resultant osmotic imbalance disrupts epithelial cell membranes (Gill et al. 1992; Knowles 1994).

The crystal structure of CrylAa has been solved (Grochulski et al. 1995) and shown to have three domains. Domain I is a bundle of seven antiparallel a-helices, domain II is three antiparallel (3-sheets arranged in a (3-prism fold (Shimizu and Morikawa 1996), and domain III consists of two twisted antiparallel (3-sheets. Domains II and III may be involved in binding midgut receptor molecules of susceptible insects. Domain II shares similarities to immunoglobulin antigen-binding sites and the plant lectin jacalin (Ge et al. 1989;

Sankaranarayanan et al. 1996). Jacalin binds carbohydrates via its p-prism fold (Knowles et al. 1984). Similarities suggested domain II may be involved in binding, and was supported by site directed mutagenesis experiments (see Schnepf et al. 1998 pg. 786 for references).

Domain III involvement was indicted by mutagenesis experiments of Cry1 Ac that decreased toxicity toward H. virescens (Schnepf et al. 1990; Ge et al. 1991) and M. sexta (Aronson et al. 1995). Domain I inserts into midgut membranes and forms and ionic channel (Schnepf et 23 al. 1998). Mutagensis of CrylAb and Cry1 Ac domain I decreased toxicity toward M. sexta

(Wu and Aronson 1992; Chen et al. 1995) and (Aronson et al. 1995) showed irreversible binding was disrupted.

Molecular Mechanism of Resistance

Native Bt toxins are translated as protoxins and require three steps to achieve insect toxicity, 1) peptide solubility, 2) conversion of protoxin to active toxin, and 3) binding of receptor molecules along the midgut epithelium prior to membrane insertion (Schnept et al.

1998). The initial step in the pathogenic mechanism is Bt protoxin solubilizaton within appropriate midgut environments. Ingestion by insects with a midgut pH near the isoelectric point (pi) of a Bt toxin may cause precipitation or denaturation, making subsequent downstream steps impossible. Thus, the different midgut pH levels between Coleoptera

(6.5), Lepidoptera (10 to 11) and Diptera (7.0) may influence target range of insects (Beegle and Yamamoto 1992). Proteolytic activation of Bt protoxin, requires endogenous proteases in the midgut lumen (Rukmini et al. 2000) and leaves lie29 and Arg601, respectively, at N- and

C-termini of the toxin. Lastly, active toxins bind receptors located on the luminal side of midgut epithelium, with candidate proteins being cadherin (Gahan et al. 2001), aminopeptidase (APN; Knight et al. 1994), and alkaline phophatase (Jurat-Fuentes and

Adang 2004). Van Rie et al. (1989), Wolfersberger (1990), and Garczynski et al. (1991) showed a lack of correlation between toxin binding affinity to midgut receptors and insecticidal activity. Irreversible toxin binding to receptors occurs only after insertion into cell membranes, and this step was determined the main kinetic determinant and critical step for toxicity (Ihara et al 1993; Liang et al. 1995; Wolfersberger et al. 1986). Furthermore, 24 toxin binding might be mediated by glycosylation state midgut epithelial receptors influenced directly by (3-1,3-galactosyltransferases (Griffitts et al. 2001, 2003).

The crystalline Bt CrylAb gene was incorporated into maize (Koziel et al. 1993) and other crop plant germplasm by transgenic technology (Frutos et al. 1999). Commercial

CrylAb toxin expressing maize hybrids were available since 1997 (Rice and Pilcher 1998) and currently planted on 29% of U.S. com ground (22.9 million acre; USDA-NASS 2004).

Transgenic CrylAb coding sequences have been truncated (3' and 5') and codons altered from the native subsp. kurstaki gene. Two copies of a 648 amino acid encoding CrylAb gene is expressed by Novartis commercial lines by fusion to a phosphoenolpyruvate carboxylase (PEPC) promoter, and represents a truncated version of the native 1155 amino acid CrylAb. 28-N and 31-C terminal residues must still be proteolytically removed from the 648 residue truncated transgenic expressed CrylAb prior to activation (Tappeser 1997).

Requirement in other transgenic com lines remains unknown, but Li et al. (2005) suggested little cross resistance between native and transgenic toxins from an O. nubilalis colony showing suppressed trypsin transcript levels (discussed below).

Transgenic CrylAb coding sequences have been truncated (3' and 5') and codons altered from the native B. thuringiensis subsp. kurstaki gene (Vaeck et al. 1998), which might affect solubility or proteolytic activation requirements by endogenous proteases in susceptible insect midguts (Schnept et al. 1998; Rukmini et al. 2000). Difference between activation requirements of truncated and native toxin was shown for a Dipel® native Bt formulation resistant O. nubilalis colony (Li et al. 2005). A 2.7 to 3.8-fold reduction in trypsin T23 transcript level in the Dipel® resistant colony offered a 254-fold increase in resistance to CrylAb protoxin compared to a susceptible strain, but only a nearly 12-fold 25 increase from active toxin. Therefore, resistance to transgenic toxins likely will evolve at the receptor binding, membrane insertion, or toxin degradation (by chymotrypsin) steps.

Lepidopteran Proteases and Bacillus thuringiensis Toxin Resistance

Trypsin-like proteases cleave following highly basic residues (Arg and Lys), and are determinants of B. thuringiensis protoxin activation (Haider and Eller 1989; Oppert et al.

1997; Rukmini et al. 2000; Li et al. 2004 and 2005). Midgut protease expression profiles vary during larval development that decrease proteolytic activation of Cryl protoxins among late instar Spodoptera littoralis compared to earlier instars (Keller et al. 1995). Reduced trypsin-like protease activity was observed among Cryl Ac resistant Plodia interpunctella

(Oppert et al. 1996) and attributed to lower trypsin transcript levels by quantitative RT-PCR

(Zhu et al. 2000b). Additionally, plant expressed trypsin inhibitors function as larval anti- feeding agents and defensive mechanisms that suppress activity of phytophagous insects.

Soybean trypsin inhibitor was shown to differentially affect protease mRNA transcript levels, revealing presence of trypsin inhibitor sensitive and insensitive gene expression (Brown et al.

1997; Mazumdar-Leighton and Broadway 2001a; Mazumdar-Leighton and Broadway

2001b). No analogous studies have been conducted using corn trypsin inhibitor compounds.

Evidence suggests the presence of a model of CrylAb activation, with protoxin cleavage dependent upon 1) larval development stage, 2) maize genetic background of potentially different constituent of trypsin inhibitors, and 3) O. nubilalis larval genetic background of differentially regulated trypsins. Developmental and genetic feedback mechanisms may modulate midgut protease expression that in turn might affect level of CrylAb protoxin activation, contributing to CrylAb resistance. 26

Chymotrypsin-like proteases cleave following aromatic residues (Trp, Tyr and Phe), and may degrade active (trypsinized) Cry toxins, rendering them nontoxic to susceptible insects (Yamagiwa et al. 1999; Miranda et al. 2001). Helicoverpa armigera chymotrypsins were implicated in breakdown of active Cry3A toxins (Carrol et al. 1997), as well as CRY4A by Culexpipiens (Yamagiwa et al. 1999). Native CRYIAb tolerance of Spodoptera frugiptera might lie with its high active toxin degradation rate compared to more susceptible

Manduca sexta larvae (Miranda et al. 2001). Although not implicating chymotrypsins,

Keller et al. (1996) also showed increased rates of CrylC toxin degradation in late instar

Spodoptera litoralis was due to enhanced serine protease activities.

Lepidopteran Midgut Receptors and Bacillus thuringiensis Toxin Resistance

Bacillus thuringiensis toxins may bind ./V-acetyl-D-galactosamine (GalNAc) modified ligands located on the luminal side of midgut epithelial cells (Knowles et al. 1991; Masson et al. 1995). Theoretically, many unrelated midgut receptor proteins may share similar carbohydrate motifs that are bound by activated Cry toxins. A M. sexta midgut membrane- bound cadherin-like glycoprotein was isolated (Vadlamudi et al. 1993) and shown to bind activated Cryl A toxins (Francis and Bulla 1995). Cadherin-like cDNAs subsequently were isolated from Lepidoptera M. Sexta (Vadlamudi et al. 1995), Bombyx mori (Nagamatsu et al.

1998), H. virescens (Gahan et al. 2001), Pectinophora gossypiella (Morin et al. 2003), and O. nubilalis (OnBt-Ri, Flanagan et al. 2005; Coates et al. 2005a). Cadherin function as a candidate midgut Bt receptor was shown by a transposon insertion-mediated cadherin knockout that reduced Cryl Aa toxicity in H. virescens (Gahan et al. 2001), and linkage of

Cryl Ac resistance to segregation of three P. gossypiella cadherin alleles (rl, r2, and r3\ 27

Morin et al. 2003). These resistant alleles also were observed in independent Cryl Ac selected colonies originally collected from Arizona (Tabashnik et al. 2004). Two putative

Cryl A binding sites were identified on M. sexta cadherin Bt-Ri peptide,

865NITIHITDTNN875 (toxin binding region 1, TBR1; Gomez et al. 2001, 2002a, 2002b) and

1331IPLPASILTVTV1342 (TBR2; Gomez et al. 2003). A third binding domain was found in the M. sexta cadherin repeat 12 (CR12), spanning amino acids 1363 to 1464. Similar to

GABA and acetylcholinesterase chemical pesticide binding site mutations in resistant alleles

(ffrench-Constant et al. 1993; Mutero et al. 1994), point mutations in proposed cadherin

CrylAb binding sites have been associated with resistant phenotypes (Xie et al. 2005). Two critical H. virescens cadherin CR12 amino acid positions, Leu1425 and Phe1429, cause loss of

Cryl A toxin binding is substituted with charged amino acids. Xie et al. (2005) further suggest L1245R mutant alleles may switch to resistant alleles by single point mutation CTG to CGG (Leu1425 to Arg1425). Coates et al. (2005a) developed molecular assays to detect single nucleotide polymorphisms (SNPs) within TBR1 (861D1EIEIIDTNN871) and TBR2

(1328IPLQTSILV VT[I/V]N1340) of the O. nubilalis cadherin gene. The single nonsynonymous mutation in toxin binding regions (I or V1339) was also present in a Chilo suppressalis cadherin, and assumed not to affect regional charge or peptide structure because alternate amino acids both have short chain aliphatic side chains (Coates et al. 2005a). The analogous CR12 domain identified by (Hua et al. 2004) is present from O. nubilalis cadherin amino acid positions 1356 to 1457, and critical amino acids in the domain are Leu/Phe1414 and Met1418. Consensus CR12 was not predicted from O. nubilalis cadherin sequences

(Coates et al. 2005a; Flannagan et al. 2005), but the third putative binding region is within 28

the membrane proximal domain. Mutations among O. nubilalis cadherin alleles are yet to be

correlated with differing Cry toxin resistance phenotypes.

Additionally, a glycosyl-phosphatidylinositol (GPI) anchored aminopeptidase isoform

with four potential 7V-glycosylation sites bound B. thuringiensis Cryl toxins in vitro (Knight

et al. 1994; Jenkins and Dean 2001). cDNAs encoding candidate Cryl A-binding APNs were

isolated from M. sexta (Knight et al. 1995; Denlof et al. 1997), H. virescens (Gill et al. 1995;

Lou et al. 1997), Lymantria dispar (Vadlamudi et al. 1995; Lee et al. 1995; Garner et al.

1999), B. mori (Yaoi et al. 1997; Hua et al. 1998; Nagamatsu et al. 1998; Nakanishi et al.

2002), P. interpunctella (Zhu et al. 2000a), Plutella xylostella (Denlof et al. 1997; Nakanishi

et al. 2002), Epiphyas postvittana (Simpson and Newcombe 2000), and Helicoverpa

punctigera (Emmerling et al. 2001). A B. mori APN deletion mutation encoding a 274

amino acid region was shown to bind Cryl A toxins (Yaoi et al. 1999), and Nakanishi et al.

(1999) further refined the APN toxin-bind domain to a 64 amino acid stretch of the P.

xylostella APN. Role of APN in Cryl Ac toxicity was demonstrated by transcript knockdown

using RNA interference (RNAi) in Spodoptera litura (Rajagopal et al. 2002), and removal of

conserved N-glycosylation sites by block deletion in B. mori APN gene (Nakanishi et al.

1999). The Bt toxin domain III may interact with GalNAc moieties of APN (Jenkins et al.

1999, 2001), which is enhanced by association with cell membrane phosphatiylcholine

(Sangadala et al. 2001).

Cryl toxin receptors from 60- to 80-kDa were described from H. virescens (Jurat-

Fuentes and Adang 2001), M. sexta (Garczynski et al. 1991), and P. interpunctella

(Mohammad et al. 1996). Proteomic analysis identified an approximate 65-kDa alkaline phosphatase, a third class of lepidopteran midgut receptor peptide (McNall and Adang 2003). 29

The receptor was a 63- and 68-kDa GPI-anchored membrane bound alkaline phosphatase with terminal acetylgalactosamine ^-oligosaccharide modifications (GalNAc) added posttranslationally. Cryl Ac bound to terminal GalNAc moieties on the II. virescens alkaline phosphatase. Moreover, the Cryl Ac resistant H. virescens strain YHD2 showed a 3-fold decrease in alkaline phosphatase activity compared to susceptible controls (Jurat-Fuentes and

Adang, 2004), and assumed to correlate with decreased levels of the epithelial membrane- bound enzyme.

Lipid rafts refer to collections of peptides and lipids (cholesterol) in the fluid membrane of midgut epithelial cells. GPI-anchored proteins are preferentially soluble in lipids contained within the rafts (Cameos et al. 1993). Therefore cadherin, aminopeptidase, and alkaline phosphatase receptors may congregate in proximity to one another within these rafts (Sangadala et al. 1994; Zhan et al. 2002), and spatially associate making contacts with

Cry toxins that strengthen proximal membrane contacts prior to insertion. This receptor- binding and membrane insertion point may be the critical step in determination of transgenic

Cry toxicity.

Unrelated midgut receptor proteins may share similar carbohydrate motifs bound by

Bt toxins. Altered expression of posttranslational modifiers that affect carbohydrate

(GalNAc) attachment may represent a general mode of resistance affecting multiple receptors

(Griffitts et al. 2001, 2003). Nematicidal toxins CrySB and Cry14A share structural homology and invoke similar physiological response as commercially used Cryl A toxins, suggesting similar mode of action (Griffitts et al. 2001). Caenorhabditis elegans is a model organism with extensive genomic information available (The C. elegans Sequencing

Consortium 1998). Gut cells from five recessive C. elegans Bt resistance (bré) mutants 30 failed to take up CrySB and Cryl4A toxins and evaded lysis, that likely was due to lack of membrane pore formation (Maroquin et al. 2000; Griffitts et al. 2001). Complementation mapping identified a C. elegans chromosome region containing a putative (3-1,3- galactosyltransferase family 5 member (|33GalT5; Amado et al 1999; Griffitts et al 2001;

Hennet 2002) with homology to Drosophila melanogaster BRAINIAC (Panin and Irvine

1998; Bruckner et al. 2000). Bre-5 and brn were shown to include #-acetylglucosaminyl transferase activity in C. elegans (Griffitts et al. 2003) and D. melanogaster (Muller et al.

2002), respectively, that may add terminal ^-acetylgalactosamine (GalNAc) to oligosaccharides or lipids (Hennet 2002). The bre mutants demonstrated lost or reduced function of posttranslational modification pathways could mediate resistance in nematodes.

Structural similarities between insect and nematode-specific toxins, and requirements for carbohydrate binding prior to pore formation in Lepidoptera suggest potential for a glycosylation pathway-mediated Bt resistance mechanism in insects (Griffitts et al. 2001;

2003).

Insights into the Ostrinia nubilalis Molecular Bt Resistance Mechanism

Trypsin and chymotrypsin-like protease activities were detected from alkaline midgut juice of 5th instar O. nubilalis (Houseman et al. 1989; Huang et al. 1999c). The effects of protease activity on Bt protoxin activation were observed in several studies. First, midgut extracts from the O. nubilalis KS-SC strain with 40 to 70-fold resistance to a Dipel® Bt formulation (Huang et al. 1997) had reduced trypsin-like protease activity and 35% decreased capacity to hydrolyze CRYIAb protoxin compared to susceptible controls (Huang et al. 1999c). Li et al. (2004) corroborated these results by showing a reduced rate of toxin 31

activation in the KS-SC strain was correlated with decreased trypsin-like protease activity. A

follow-up study by Li et al. (2005) demonstrated a 2.7 to 3.8-fold reduction in trypsin T23

transcript level in Dipel® resistant compared to susceptible individuals using real-time PCR.

The Dipel® resistant phenotype in the KS-SC showed an approximate 11-fold resistance toward CrylAb protoxin compared to a trypsinized version, suggesting lower resistance to transgenic CrylAb that likely is expressed in active form (Vaeck et al. 1987; Tappeser 1997).

Genomic markers for association of alleles at the T23 locus with segregating Bt resistance phenotypes were developed in chapter 7 (Coates et al. 2005d).

Binding of CrylAb and Cryl Ac toxins appeared to share receptor-binding sites in the

O. nubilalis midgut, and these toxins may interact with two different receptors (Denlof et al.

1993). Moreover, CrylAb binding was approximately 10-fold strong than Cryl Ac. In competition assays, Hua et al. (2001) found Cry IF had low affinity for the CrylAb receptor, whereas Cry9A and Cry9C toxins may have independent binding receptors. Far western blot using indicated CrylAb peptide binding occurred at strongly to 220, 154, and 145 kDa O. nubilalis midgut receptors, and weakly to a 167-kDa peptide. Immunoblotting with specific antibodies demonstrated cadherin-like (220-kDa) and aminopeptidase isoforms (167, 154 and

145-kDa) receptors may function as CrylAb toxin receptors in O. nubilalis (Hua et al. 2001).

In contrast, Cryl Ac and Cryl F toxins associated with the 220-kDa cadherin and 154-kDa aminopeptidase peptides. Although not addressed by Hua et al. (2001), approximately 100 and 8 5-kDa peptides were bound (weakly) by CrylAb and Cryl Ac, and might correspond to alkaline phosphatases (Garczynski et al. 1991; Jurat-Fuentes and Adang 2001).

Contradictory results by Flanagan et al. (2005) suggest CrylAb binds three cadherin (OnBt-

R,) isoforms of 220, 170, and 160-kDa, but offered no specific explanation for differences 32 with previous studies. Transformation of CrylAb insensitive Spodoptera frugiperda (Sf9) cells with a pFastBac construct containing OnBt-Ri demonstrated susceptibility was attained at 0.1 fj,g CRYlAb/ml. Reduction in size of the 190-kDa S19 cell line expressed cadherin compared to native OnBt-Rj was assumed due to lack of post-translation modification machinery in S19 cells. Since proper glycosylation, including GalNAc addition, may not take place in S19 cells, and aminopeptidase were not included in the studies, results by Flanagan et al. (2005) suggest presence of a cadherin peptide is sufficient to cause membrane liability to CrylAb toxin. Analogous transformation studies with aminopeptidase isoforms will shed light into the necessity of cadherin OnBt-Ri in the toxin binding mechanism.

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CHAPTER 2. PARTIAL MITOCHONDRIAL GENOME SEQUENCES OF OSTRINIA NUBILALIS AND OSTRINIA FURNICALIS

Brad S. Coates, Douglas V. Sumerford, Richard L. Hellmich, and Leslie C. Lewis

Paper published: International Journal of Biological Sciences, 1:13-18, 2004

Abstract

Contiguous 14,535 and 14,536 nt near complete mitochondrial genome sequences respectively were obtained for Ostrinia nubilalis and Ostrinia furnicalis. Mitochondrial gene order was identical to that observed from Bombyx. Sequences comparatively showed 186 substitutions (1.3% sequence divergence), 170 CDS substitutions (131 at 3rd codon positions), and an excess of transition mutation likely resulting by purifying selection (d^/dg

= G) = 0.15). Overall substitution rates were significantly higher at 4-fold (5.2%) compared to 2-fold degenerate codons (2.6%). These are the 3rd and 4th lepidopteran mitochondrial genome reference sequences in GenBank and useful for comparative mitochondrial studies.

1. Introduction

Mitochondrial genomes of 16 insect species are completely sequenced and published with a majority from the order Diptera; D. yakuba (Clary and Wolstenholme 1985), A des gambiae (Beard et al. 1993), Anopheles quadrimaculatus (Mitchell et al. 1993), D. melanogaster (Lewis et al. 1995), Ceratitis capitata (Spanos et al. 2000), Cochliomiyia hominivorax (Lessinger et al. 2000), D. simulins (Balard 2000), and Bactrocera oleae (Nardi et al. 2003). Complete sequences also have been published from a hymenopteran, Apis mellifera (Crozier et al. 1993), an orthopteran, Locusta migratoria (Flook et al. 1995), a phthirapteran H. macropus (Shao et al. 2001), thysanuran, T. inaginis (Shao and Baker 49

2003), a hemipteran Triatoma dimidiata (Dotson and Beard 2003), coleopteran Crioceris duodecipunctata (Steward and Beckenback 2003), and lepidopterans Bombyx mori and B. mandarina (Yukuhiro et al. 2002).

Larvae from corn borer species Ostrinia nubilalis and Ostrinia furnicalis

(Lepidoptera: Crambidae) are pests of agricultural crop plants and cause major crop production losses (Mason et al. 1996; Showers 1993). Ostrinia nubilalis and O. furnicalis are sister species (Mutuura and Munroe 1970), with difference residing in female O. nubilalis and O. furnicalis emission of E- and Z- stereoisomers of All- (Klun and Huettel 1988), and

A12-tetradecenyl acetates (Huang et al. 1998), respectively. The pheromone binding protein gene sequences showed little nucleotide variance between O. nubilalis and O. furnicalis

(Willet and Harrison 1999), and 7 allozyme markers indicated a high similarity between

Chinese populations of O. nubilalis and O. furnicalis suggesting recent speciation (Wang et al. 1995). Similarly, mitochondrial cytochrome c oxidase subunit II (coxl) gene alignment estimated 1.63% interspecies divergence (Kim et al. 1999). The present study compares

GenBank annotated mitochondrial genomes from O. nubilalis (accession AF442957) and O. furnicalis (AF467260).

2. Materials and methods

2.1 Samples and amplification

A single bivoltine female Z-pheromone race O. nubilalis adult was collected from the

Iowa State University Uthe Farm, Ames, Iowa, USA. One adult multivoltine O. furnicalis female of indeterminate pheromone composition collected from Hengshui, Hebei Province,

China was contributed by Dr. Wang Zhen-ying, Institute of Plant Protection, Chinese 50

Academy of Agricultural Sciences, Beijing, China. DNA extractions used Qiagen DNeasy kits (Qiagen, Valencia, CA).

Primers combinations TY-J-1460 with TK-N-3785, J-11545 with N-12854, and Nl-J-

12585 with SR-N-14588 (Simon et al. 1994) were used to PCR amplify fragments 2, 9, and

10 (Fig. 2.1). Bombyx mori (GenBank:AF149768 and AY048187) andD. yakuba (GenBank:

MIDYRRN; Clary and Wolstenholme 1985) mitochondrial genomes were aligned using

AlignX software (Informax, San Francisco, CA) to identify regions of sequence similarity, from which regions PCR primers were designed to amplify remaining fragments using

PrimerS (Rozen and Skaletsky 1998). All PCR reactions were performed in a 50 pi volume with 1.7 U of Tli polymerase (Promega Corp., Madison, WI), 100 ng of DNA, 5 (j,l 10X thermal polymerase buffer (Promega), 2.5 mM MgCh, 200 (iM dNTPs, and 20 pmol of each primer. Fragments 1, and 3 to 8 were amplified on a PTC-100 thermocycler (MJ Research,

Watertown, MA) with denaturation at 95°C for 2 m, followed by 40 cycles at 94°C for 30 s,

50 to 54 °C for 40 s, a 2.5 °C/s ramp for +15 °C, and 70 °C for 1.5 to 3 m depending on fragment length. Fragments 2, 9, and 10 were amplified by denaturing template at 95°C for 2 m, followed by 40 cycles at 94°C for 30 s, 44 °C for 1 m, a 2.0 °C/s ramp for +23 °C, and 70

°C for 3 m.

2.2 DNA sequence and analysis

PCR reaction products for fragments 1 to 10 were purified using Qiaquick PCR purification columns (Qiagen), and diluted to 2.5ng/p,l/100 bp of product length. Sequencing was performed in duplicate at the DNA sequencing core facility at Iowa State University, 51

Ames, IA. Overlapping fragments were assembled into a single contiguous sequence using

Contig Express software (Informax). Ostrinia nubilalis and O. furnicalis mitochondrial

genome sequences were aligned with B. mori (GenBank AF149768, and GenBank

AY048187) using AlignX software (Informax), and gene features were annotated using

Vector NTI 7.0 (Informax). Contiguous mitochondrial DNA sequence of 14535 and 14536 nt were respectively submitted to GenBank for O. nubilalis (AF442957) and O. furnicalis

(AF467260).

Substitution rate and transition/transversion ratio for Ostrinia mitochondrial DNA sequences were calculated with MacClade 4.03 (Maddison and Maddison 2001). Twenty one tRNA gene structures were predicted with M-fold 3.1 (Zuker et al. 1999), and viewed

using RNAviz 2.0 (De Rijk and De Wachter 1997). Codon usage was evaluated by the

Countcodon program version 4 (http://www.kazusa.or.jp/codon/ countcodon.html). Average per site rates of synonymous (d$) and nonsynonymous nucleotide substitution (d%) were calculated according to (Nei and Gojobori 1986) using MEGA (Kumar et al. 1993).

3. Results and discussion

3.1 Ostrinia mitochondrial genomes

Contiguous O. nubilalis (GenBank accession: AF442957) and O. furnicalis

(AF467260) mitochondrial genomes were assembled from overlapping PCR product sequence (Fig. 2.1). Each GenBank record includes 13 open reading frames (ORFs), a large ribosomal RNA (rrnL) gene, 21 tRNAs, and part of trnM and small ribosomal RNA (rrnS) genes (Fig. 2.1). Gene order and orientation were identical to Drosophila (Clary and

Wolstenholme 1985, Lewis et al. 1995), except for translocation of trnM to a position 52 preceding trnl as was observed in Bombyx (Yukuhiro et al. 2002). Major strand of O.

(41.3% A, 38.8% T, 8.0% G, and 11.8% C; 80.2% AT) and (41.5% A,

38.9% T, 7.9% G, and 11.7% C; 80.4% AT) showed a bias toward A and T nucleotides that is typical of insect mitochondrial genomes (Boore 1999).

The O. nubilalis and O. furnicalis mitochondrial genomes have 3731 codons; 3718 amino acid encoding and 13 termination codons (Table 2.1). Codons had a prevalence of A and T in 3rd positions and bias may reflect selection for optimal tRNA use (Xia 1996), speed of genome replication, genome bias, or DNA repair efficacy (Table 2.2). The O. nubilalis and O. furnicalis mitochondrial peptides comparatively showed 24 predicted amino acid changes (24 of 3718; 0.646%; peptide similarity = 99.22%, and identity ~ 99.78%) (Li 1997).

All ORFs were initiated by ATA or ATT codons, except coxl. Initiation of coxl translation is ambiguous, but may occur by a TATT AG sequence in O. nubilalis and O. furnicalis, that is similar to TTTTAG in the B. mori. Hexanucleotides, initiation signals TATCTA from

Penaeus monodon (Wilson et al. 2000), or ATTTAA from A. gambiae (Mitchell et al. 1993),

A. quadrimaculatus (Lewis et al. 1995) and C. capitata Spanos et al. 2000 have been proposed. Alternatively, an ATAA tetranucleotide sequence was predicted to initiate coxl translation in Drosophila, L. migratoria (Flook et al. 1995), and Daphnia pulex (Crease

1999). Termination codons were either TAA or TAG in O. nubilalis and O. furnicalis, except for cox2 and atp6 that have incomplete stop codons T and TA, respectively.

Incomplete stop codons may become function after polycistronic transcript cleavage and polyadenylation mechanisms (Ojala et al. 1980; Anderson et al. 1981).

Complete nucleotide sequence was obtained for 21 O. nubilalis and O. furnicalis mitochondrial tRNAs. Seven substitutions were observed, and 0.49% sequence divergence 53 was estimated from 1429-shared sites. Insertion-deletion (indel) mutation occurred in loop structures of trnA, trnD, trnG, and trnT, and, except for trnR, did not affect predicted two- dimensional tRNA structures (Fig. 2.2). Variable mitochondrial tRNA loops in Bombyx were assumed not to affect biological function (Yukuhiro et al. 2002). The complete rrnL gene sequence was 1339 nt for O. nubilalis and O. furnicalis, and alignment comparatively showed a single C to T transition. A partial rrnS sequence was obtained from O. nubilalis

(434 nt) and O. furnicalis (435 nt), and comparatively showed a single nucleotide deletion.

3.2 Nucleotide substitution pattern

A 14543 nt consensus mitochondrial genome alignment identified 186 substitutions between O. nubilalis and O. furnicalis: 138 transition (ts) and 48 transversion (tv) mutations

(ts:tv = K S 2.88). This ratio deviated significantly from neutral expectation (1:2; %2 =

141.447, d.f. = 1 ,P< 0.001), indicating evolutionary pressures are acting upon O. nubilalis and O. furnicalis mitochondrial genomes. Excess transition mutation also was reported between D. melanogaster subgroup members (K= 761/180 S 4.23) and attributed to non- neutral evolutionary forces or population effects (Balard 2000).

Additionally, mitochondrial protein coding sequences (CDS) comparatively showed

170 substitutions between O. nubilalis and O. furnicalis', 131 at 3rd codon positions. The ratio of the rate of nonsynonymous changes at nonsynonymous sites (dN) to synonymous changes at synonymous sites (d$) in Ostrinia ORFs indicated a 7-fold excess of silent mutation (dN/ds = co = 0.15) (Nei and Gojobori 1986). High peptide similarity (= 99.22%) may reflect regency O. nubilalis and O. furnicalis speciation, but effects of purifying selection can be inferred since synonymous substitutions are very prevalent. Alternatively, 54 similar environmental selection after speciation could lead to peptide conservation co- occurring with a background of random genetic drift at neutral nucleotide positions. The

observed mutation rate at Ostrinia 4-fold degenerate codons (|i4_r0id = 5.22%) was significantly higher than at 2-fold degenerate codons (|j.2-foid = 2.60%; %2 = 35.157, d.f. = 1, P

< 0.001). Results suggest a greater susceptibility of 4-fold degenerate codons to synonymous substitution.

3.3 Divergence time estimates

The divergence time between O. nubilalis and O. furnicalis mitochondrial was estimated by assuming a linear rate of substitution in short-term evolution (molecular clock)

(Zuckerland and Pauling 1965) of 2% per million years (Powell 1986). Nucleotides in rRNA and tRNA may lack independence due to structural dependence, and purifying selection may act at 1st and 2nd codon positions. The intergenic sequence (IGS) and 3rd codon positions only sites that are nearly neutral. IGS region and 3rd codon positions showed 3.54% nucleotide difference between O. nubilalis and O. furnicalis, indicating that speciation occurred 1.8 mya (Powell 1986). Alternatively, 3rd position and IGS region data give a pairwise genetic distance of 0.3284 ± 0.0348 using the Kimura-2-parameter model (Kimura

1980). Estimates of 0.1 distance unit (D) per 1.0 myr (Osawa et al. 1999) suggest divergence at 3.3 mya. These molecular-based divergence time estimates are supported by highly similar morphology of O. nubilalis and O. furnicalis (Mutuura and Munroe 1970). 55

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Figure 2.1. Ostrinia mitochondrial genome map of sequenced regions. Protein coding genes represented by arrows indicating direction with left-facing arrows on major strand.

The tRNA genes are labeled by single letter codes and * indicating coding sequence on minor strand. Underscores indicate positions of ten overlapping PCR amplified genome fragments.

Ostrinia physical mitochondrial genome map R F I C ATP8 \ I / I CytB I ND1 Î IrRNA "-srRNA

A HE PCR y I fragments

Figure 2.2. Predicted tRNAARG secondary structures. A) O. nubilalis and B) O. furnicalis trnR.

A) B)

Acceptor Acceptor

-60 WC arm 60 C C U C A c c u c nP D-loop A - - - - 50_; A G A G U A GAG A U U (y)A A GEB-10 © AQG-10 A S U A U U U G Variable U GC- 4 A Variable C-20 loop u u °-G A loop A u AA u u A u u A u A U A A U 0 = Substitution A U C U c U 30 A 0- Insertion/ u 30 A u Deletion UcG Anticodon Anticodon 59

Table 2.1. Mitochondrial codon usage table. 3718 amino acid residues and 13 nonsense codons among protein coding regions from each O. nubilalis (On) and O. furnicalis (Of)

using the invertebrate mitochondrial genetic code.

Codon On Of Codon On Of Codon On Of Codon On Of UUU-Phe 347 354 UCU-Ser 93 95 UAU-Tyr 175 170 UGU-Cys 29 30 UUC-Phe 31 28 UCC-Ser 12 10 UAC-Tyr 12 16 UGC-Cys 2 1 UUA-Leu 459 454 UCA-Ser 94 94 UAA-Ter *12 *12 UGA-Trp 89 89 UUG-Leu 15 17 UCG-Ser 5 4 UAG-Ter 1 1 UGG-Trp 5 5

CUU-Leu 20 22 CCU-Pro 60 60 CAU-His 55 58 CGU-Arg 14 14 CUC-Leu 0 1 CCC-Pro 8 7 CAC-His 7 4 CGC-Arg 1 1 CUA-Leu 32 32 CCA-Pro 55 52 CAA-Gln 61 61 CGA-Arg 33 33 CUG-Leu 0 0 CCG-Pro 1 2 CAG-Gln 3 3 CGG-Arg 3 3

AUU-lle 449 455 ACU-Thr 71 72 AAU-Asn 232 234 AGU-Ser 23 25 AUC-Ile 28 28 ACC-Thr 14 10 AAC-Asn 24 22 AGC-Ser 2 2 AUA-Met 262 265 ACA-Thr 71 75 AAA-Lys 92 94 AGA-Ser 90 89 AUG-Met 26 20 ACG-Thr 1 0 AAG-Lys 9 8 AGG-Ser 0 1

GUU-Val 72 70 GCU-Ala 70 71 GAU-Asp 59 59 GGU-Gly 56 56 GUC-Val 1 2 GCC-Ala 9 8 GAC-Asp 4 4 GGC-Gly 1 5 GUA-Val 58 62 GCA-Ala 46 47 GAA-Glu 65 62 GGA-Gly 117 115 GUG-Val 6 1 GCG-Ala 2 1 GAG-Glu 8 10 GGG-Gly 29 25 Includes stop codons from cox2 (T) and atpô (TA), completed by adenylation.

Table 2.2. Nucleotide frequencies in mitochondrial genomes, partitioned among O. nubilalis (On) and O. furnicalis (Of) mitochondrial genome regions. IGS = non-coding intergenic spacer regions.

Protein Coding Sequence Imposition 2nd position 3rd position rrnL rrnS tRNAs IGS % nt On Of On Of On Of On Of On Of On Of On Of %AT 74.4 74.5 70.4 70.5 92.8 93.3 84.9 85.0 82.3 82.8 82.2 82.1 91.9 93.4 % GC 25.6 25.5 29.6 29.5 7.2 6.7 15.1 15.0 17.7 17.2 17.8 17.9 8.1 7.6 60

CHAPTER 3. GEOGRAPHIC AND VOLTINISM DIFFERENTIATION AMONG NORTH AMERICAN OSTRINIA NUBILALIS (LEPIDOPTERA: CRAMBIDAE) MITOCHONDRIAL CYTOCHROME C OXIDASE HAPLOTYPES

Brad S. Coates, Douglas V. Sumerford, Richard L. Hellmich, and Leslie C. Lewis

Paper published: Journal of Insect Science, 4:35, 2004

Abstract

DNA sequence of European corn borer, Ostrinia nubilalis (Hubner), mitochondrial cytochrome c oxidase I {coxl) and II {cox2) genes was characterized and used for population genetic analysis. Twenty-six point mutations were identified from a 2,156 bp DNA sequence alignment. Frequency of polymorphic coxl Ddel and Haelll, and cox2 Sau3Al and Mspl restriction sites were determined from 1,414 individuals by polymerase chain reaction restriction fragment length polymorphism. Ten haplotypes were observed. A single haplotype was present among 90% of individuals examined, and a Haelll haplotype was not present in samples from the Atlantic coast. Significant genetic differentiation existed between Atlantic coast and midwestem United States samples, and between sympatric uni- and bivoltine ecotypes. These genetic markers identify regional and ecotype differences in the North American O. nubilalis population.

Introduction

Population structure and mating barriers affect rates of gene flow within a species

(Mutebi et al. 2002). European com borer (ECB), Ostrinia nubilalis (Hubner), is an introduced insect pest of agricultural crops in North America (Mason et al. 1996). Ostrinia nubilalis is endemic to Europe and western Asia, and by 1917 was observed in the eastern 61

United States, migrated westward, and reached North Dakota by 1950 (Chiang 1972;

Showers 1993). The North American O. nubilalis population shows phenotypic variation in pheromone production and perception, and generation number (voltinism; Showers 1993;

Mason et al. 1996).

The female O. nubilalis pheromone, 11-tetradecenylacetate, is produced in stereoisomeric forms (E and Z) and in varying blend ratios (Roelofs et al. 1972; Klun et al.

1973). Two ecotypes, the ^-strain (New York type) and Z-strain (Iowa type), use 98:2 and

1:99 mixtures of (E)- and (Z)-l 1-tetradecenyl acetates, respectively (Glover et al. 1987). The

Z-pheromone-emitting and responding populations are distributed in the central and eastern

United States and southern Canada (Showers et al. 1974), whereas the £-strain inhabits the northeastern United States and Quebec (McLeod et al. 1979). The distribution of pheromone races overlap in the eastern United States and intermating of pheromone races was documented (Roelofs et al. 1985; Durant et al. 1995).

Ostrinia nubilalis ecotypes, uni-, bi-, and multivoltine, differ in response to photoperiod and temperature for diapause induction, resulting in differences in degree-days required for pupation (Showers 1979). Genes for voltinism determination are male sex- linked, and show temperature- and scotophase-dependent dominance (Showers et al. 1972;

Showers 1981). Univoltine ecotypes may have a selective advantage in northern climates where short growing seasons favor a single generation, whereas in more temperate southern regions extended larval dormancy may result in increased mortality (Showers 1993). Genetic and environmental factors determine voltinism phenotypes, resulting in north-south dines that correspond to barrier latitudes that restrict univoltine ecotypes to a northern distribution

(Showers 1979; Showers 1993). Movement of bivoltine ecotypes into traditionally 62 univoltine regions without reciprocal migration supports the concept of southern barrier latitudes for univoltine migration (Chiang et al. 1965; McEwen et al. 1968). Variation in post-diapause developmental time between voltinism ecotypes (Calvin and Song 1994;

Hoard and Weiss 1995) may promote asynchrony in mating period that minimizes genetic exchange (Roelofs et al. 1985). To date, no significant genetic differences have been detected between co-existing (sympatic) voltinism ecotypes.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) evaluation of four mitochondrial DNA fragments (Marcon et al. 1999), and variation at six allozyme markers (Bourguet et al. 2000) showed no genetic differences among voltinism ecotypes. PCR-random amplification of polymorphic DNA (RAPD) markers indicated multivoltine ecotypes (> 2 generations per year) were genetically different from both uni- and bivoltine ecotypes (Pornkulwat et al. 1998), but voltinism differences were confounded by geographic variance. Coates and Hellmich (2003) showed regional variation of northern

(Minnesota) subpopulations from all other sample sites at two sex-linked microsatellite marker loci. In the current study polymorphism in O. nubilalis coxl and cox2 genes was used to investigate contributions of voltinism, pheromone race, and geographic location to

North American population structure.

Materials and Methods

Sample Preparation

Second-flight O. nubilalis adults were obtained from 14 North American locations in

(Fig. 1) light or pheromone bait traps. At Lamberton, Minnesota (MN), and Rosemount, MN season-long monitoring separated peaks of uni- and bivoltine ecotypes. Two pheromone bait 63 traps, one E- and the other Z-, were placed in proximity at Cape Elizabeth, Maine (ME) and

Oxford, ME. An ^-pheromone trap was used at the Newark, DE location. The last sample site was the Ithaca, NY colony of Z- pheromone individuals were originally collected May

1996 near Geneva, NY, or E-pheromone individuals were in continuous culture since April

1994, after initial collection from fields in Bouchville, New York (NY). The laboratory colony is maintained at the New York State Agricultural Experiment Station, Geneva, NY.

All other samples were collected in light or pheromone traps (see Fig. 1). DNA extraction was performed on individual thoraces as described by Marcon et al. (1999). DNA was suspended in 100 (il of TLE (10 mM Tris, 0.1 mM EDTA, pH 7,5), and DNA concentrations were adjusted to 50 ng/pl with TLE, and stored at -20°C.

DNA Sequencing

Mitochondrial coxl, ^r«LUUR, and cox2 gene sequences were compared among 14 individual adult com borers. PCR of individual DNA samples used 50 pi reactions with 2.5 mM MgCl], 0.8 |JM dNTPs, 10.0 pmol of each primer TY-J-1460 and TK-N-3785 (Simons et al. 1994), 400 ng of DNA, and 1.7 U of Taq polymerase (Promega, www.promega.com).

A temperature cycle of 94°C for 3 min., followed by 40 cycles of 94°C for 40 sec., 53°C for

50 sec., and 72°C for 2 min. was carried out on a PTC-100 thermocycler (MJ Research,

Watertown, MA). Purification of PCR reactions used Qiaquick PCR purification columns

(Qiagen, www.qiagen.com) according to manufacturer directions, and 200 ng used in dye- terminator cycle sequencing quick start (DTCS-quick) primer extension reactions (Beckman-

Coulter, www.beckman-coulter.com). Dye-terminator cycle sequencing (DTCS) reaction 64 products were injected at 1 lOkV for 15s onto a CEQ8000 capillary electrophoresis system

(Beckman-Coulter) and separated at llOkV for 120min. Sequence reaction data were imported into Vector NTI Suite 7.0 (Informax, www.informax.com), and individual contigs were constructed. Coxl and cox2 sequences were aligned with homologous regions the O. nubilalis (GenBank AF442957) and O. furnicalis (GenBank AF467260) mitochondrial genomes by using AlignX software (Informax; gap penalty of 5). Amino acid sequence was determined with Sequin 4.00 (http://www.ncbi.nlm.nih.gov).

PCR-RFLP Methods

Three PCR primer pairs, TY-J-1460 (Simons et al. 1994) with OnCox-D-R (5-TCCA

GGATTACCTAATTCAGCTC-3 '), OnCox-H-F (5 -CACGAGCTTACTTTAC CTCAGCA-

3') with OnCox-H-R (5 -CCAGCTAGCCCTAAGAAATGTTG-3 ), and OnCox-SM-F (5'-

GGCTA GC TGGTATACCTCGAC-3 ) with OnCox-SM-R (5 '-GGAGAGGCTCTATTTTG

TAGACTA-3') spanned polymorphic regions. All PCR amplifications used 1.5 mM MgClz,

0.5 uM dNTPs, 5 pmol of each primer, 0.225 U of Taq DNA polymerase (Promega), and 150 ng of DNA in a 12.5 pi reaction volume. Thermocycler conditions were 94°C for 2 min., followed by 40 cycles of 94°C for 30 sec., 52°C for 30 sec., and 72°C for 20 sec. Single 25 pi digests with DdeI, Haelll, Mspl, or Sau3AI including 5.0 pi of PCR product, 2.5 pi 10X buffer (Promega), 0.1 mg/pl bovine serum albumin, and 0.5 U of enzyme (Promega) were incubated at 37°C for 8 to 14 hr. Reactions were loaded on 1.0 mm by 16 cm 6% polyacrylamide (29:1 acrylamide: bisacrylamide) 0.5X Tris borate EDTA gels, and separated at 140 V for 4 h with a 25-bp step-ladder (Promega) for size comparison. Gels were stained 65 with ethidium bromide, and digital images were taken on a PC-FOTO/Eclipse documentation system (Fotodyne, Hartland, WI).

Data Analysis

Pairwise effective number of migrants (Nm) were calculated from linearized FST estimates (Slatkin, 1995) and measured between Atlantic coast and Midwest samples by using estimated F-statistic; Nm = ((1/ FsT ) - 1) (Hartl and Clark 1997). Genetic distance and analysis of molecular variance (AMOVA) were calculated as described by Weir and

Cockerham (1984) and Excoffier et al. (1992) by using Arlequin vl.l (Schneider et al. 1997).

AMOVA tested regional population subdivision (Atlantic coast verse midwestem samples) and sympatic voltinism ecotype differentiation at Lamberton and Rosemount collection sites. Haplotype relationships were investigated by phylogenetic analysis of PCR-RFLP and

DNA sequence alignment data by using parsimony which incorporated 1,000 bootstrap resampling steps performed using Seqboot, followed by Mix or DNAPars, and Consense programs in the PHYLIP package (Felsenstein 1989). A haplotype network was constructed from RFLP data using the program TCS version 1.12 (available at http://inbio.byu.edu/

Faculty/kac/crandall lab/programs. htm). Genetic differentiation by geographic distance model was tested by comparison of logio distance (km) to logio genetic distance estimates

(Slatkin 1993) using NTSYS-pc v. 1.70 (Rohlf 1992), and MXCOMP of NTSYS-pc V. 1.70 to perform Mantel tests with 1000 iterations to test significance. Three levels of comparison were made using ECB genetic distance (D) estimates 1) among Atlantic coast and 2) among midwestem sample sites, and 3) between Atlantic and midwestem sample sites. ANOVA was performed using Proc Mixed of the SAS statistical software (v. 8). 66

Results and Discussion

DNA Sequence Analysis

Mitochondrial coxl, tRNA-LeuUUR, and cox2 genes were DNA sequenced from 14 O. nubilalis moths to survey genetic variation within the species. A 2,156 bp sequence alignment showed 26 point mutations of which 7 resulted in nonsynonymous amino acid changes (Table 1). Individual mutations were rare and resided in only one or two sequences.

Coxl and cox2 gene sequences were highly similar (>99.5 and >99.2%, respectively), and no mutations were observed in tRNA-LeuUUR. Screening of 1,414 O. nubilalis moths from 15 collection sites (Fig. 1) revealed low polymorphism at DdeI, Haelll, Mspl, and Sau3Al sites, and a non-digested mitochondrial haplotype among 90% of individuals (1,267 of 1,414;

Table 2). Low O. nubilalis mitochondrial polymorphism agreed with reports by Marcon et al. (1999), and was congruent with allozyme marker (Harrison and Vawter 1977; Cianchi et al. 1980; Glover et al. 1991; Bourguet et al. 2000) and sex-linked microsatellite data (Coates and Hellmich 2003). Low mitochondrial variation may result from a genetic bottleneck at

North American O. nubilalis introduction, selection, or mitochondrial DNA fixation by genetic drift. Similar results were observed from moth species, the gypsy moth, Lymantria dispar (L.) (Harrison et al. 1983), and tobacco budworm, Heliothis virescens (F.) (Roehrdanz et al. 1994), suggesting little intraspecific differentiation within the genera.

Phylogenetic methods and haplotype networks can construct haplotypes relationships.

Parsimony trees constructed from DNA sequence (Fig. 2) or PCR-RFLP haplotypes (not shown) were inconclusive with respect to haplotype relationships. The parsimony-based phylogenetic tree using DNA sequence data produced bootstrap branch support above a 50% threshold (n = 1,000 bootstrap resample steps) at only 4 of 10 nodes. Nested clade analysis 67

(NCA; Tempieton et al. 1992) was similarly unsuitable due to generation of a circular haplotype-network (data not shown). Inability to resolve haplotype relationships using phylogenetic methods may reside in absence of sufficient differentiation among O. nubilalis coxl and coxl haplotypes (Page and Holmes 1998). .

Geographic Population Differentiation

Polymorphism was absent from Ithaca, NY laboratory colony samples, and these data were omitted resulting in 14 collection sites for analysis. An isolation-by-distance model was rejected as the basis of genetic differentiation between 14 North American O. nubilalis collection sites. Using the program NYSYS-pc v. 1.70 (Rohlf 1992), genetic distance versus geographic distance (km) matrices were plotted, and showed no correlation (R2 = 0.1132, P -

0.9759). Therefore, geographic distance between sample sites may not be correlated with genetic differentiation. Alternatively, we investigated geographic region of collection site,

Midwest or Atlantic coast, as the basis of genetic variation. A direct comparison of Haelll haplotype frequency between Midwest (5.1%; 54 of 1,055) and Atlantic coast samples

(0.0%), indicated presence of a region specific haplotype (private allele; Table 2).

Population differentiation imparted by absence of Haelll haplotypes from Atlantic coast samples was investigated using modified fixation indices (^-statistics; Weir and

Cockerham, 1984; Excoffier et al. 1992), analysis of molecular variance (AMOVA;

Excoffier et al. 1992), and genetic distance estimates (Nei 1972; Schneider et al. 1997). A comparison of midwestem and Atlantic coast Ddel, HaeIII, Mspl, and Sau3Al RFLP haplotype frequencies generated a modified fixation index, of 0.024 which indicated moderate inbreeding and significantly higher relatedness of haplotypes within each region (P 68

< 0.0001). Low but significant mitochondrial haplotype differentiation between O. nubilalis

corroborates prior studies (Harrison and Vawter 1977; Cianchi et al. 1980; Glover et al.

1991; Coates and Hellmich 2003). Analysis of molecular variance (AMOVA) comparing

midwestem and Atlantic coast haplotype frequencies indicated 1.64% of total variation

within North America might be due to regional differences (Table 3; Panel A). Presence of a single non-digesting haplotype among 90% of individuals (w+; Table 2) may contribute to

low variation between geographic regions. Absence of the Haelll haplotype from Atlantic coast samples suggest it may be the basis of the low-level geographic (regional)

differentiation, and its relative contribution was estimated by excluding it from fixation index

estimation and comparison to original values. After removal of HaeIII PCR-RFLP haplotypes, the fixation index (6^T) decreased to 0.017 (P < 0.010; data not shown),

suggesting Haelll haplotypes contributed 29% of the measurable increase in relatedness of haplotypes within each geographic region ((0.024-0.017)/0.024 ~ 0.29). These analysis and raw haplotype frequency data suggest the HaeIII haplotype might have regional geographic

specificity or reflect North American O. nubilalis population subdivision.

Genetic distance estimates from 14 O. nubilalis collections sites were generated in a

pairwise manor (Ithica, NY laboratory colony haplotypes omitted), and comparisons

supported evidence of O. nubilalis geographic differentiation previously inferred from

fixation indices and AMOVA (Table 3A). Genetic distances were estimated separately for

Lamberton and Rosemount, MN voltinism ecotypes, and Cape Elizabeth and Oxford, ME

pheromone races (i.e. treated as separate subpopulations and analyzed as separate entities;

Table 2; discussed in next section). Genetic distance estimates suggested differentiation between 16 of 153 comparisons using a Bonferroni corrected significance threshold (P < 69

0.0003; Table 4). No significant difference in genetic distance was predicted between any two collection sites or ecotypes on the Atlantic coast, suggesting greater genetic similarity within Atlantic coast regions compared to the Midwest. Geographic similarity was further investigated by one-way ANOVA comparing genetic distance (D) between Atlantic coast and midwestern samples, and resulted in significant differentiation (F = 14.53; df = 2, 150; P

< 0.0001).

Exclusion of the Haelll haplotype from Atlantic coast regions suggest 1) low frequency of Haelll haplotype migration, or 2) separate and distinct regional O. nubilalis introductions. Assuming Hardy-Weinberg equilibrium, a historic effective female migration rate (N/n = ((l/FSi) - 1); Hartl and Clark 1997) between Atlantic coast and midwestern regions was estimated at 40 to 41 female migrants per generation (using (%T = 0.024; Table

3). Migration estimates are crude due to unrealistic island model assumptions (Hartl and

Clark 1997), but suggest a historically moderate level of haplotype exchange. Migration rate estimates may be lower due to inability to differentiate many O. nubilalis mitochondrial haplotypes. The second hypothesis assumes Atlantic and midwestern differences result from separate North American O. nubilalis introductions. Records indicate O. nubilalis infestations in eastern North America near Boston (1913) and eastern New York State

(1919), and a single midwestern introduction near Lake Erie (Vinal 1917; Showers 1993). If

Boston and New York introductions populated Atlantic coast regions and the single Lake

Erie introduction populated the Midwest, present day genetic differentiation might be indicative of European founders. Alternatively, Haelll haplotype extinction in the Atlantic coast region could have resulted after introduction due to random genetic drift within a small founder population or selection within new habitats. Sampling error in the current study is 70 unlikely due to collection sizes at multiple regional locations (Fig. 1; Table 2). Additional sampling is required for follow up confirmatory studies.

Sympatric Ecotype Differentiation

Coexisting (sympatric) pheromone races were present in Oxford (site 1) and Cape

Elizabeth, ME (site 2) pheromone traps. Comparison of mitochondrial haplotypes between pheromone races showed no significant inbreeding effects within ecotype at Oxford and

Cape Elizabeth, ME locations (<%T = 0.021; P = 0.081), suggesting gene flow between pheromone ecotypes and corroboration of pheromone hybrid females observed in the field

(Roelofs et al. 1985; DuRant et al. 1995). Sympatric voltinism ecotypes also were collected from the same light trap at Lamberton (site 12) and Rosemount, MN (site 13). Comparison of haplotype frequency among sympatric voltinism ecotypes may represent a measure of ecotype variation in absence of confounding geographic effects. In contrast to pheromone ecotypes, differences were detected when haplotypes from Minnesota collection sites when separated into voltinism ecotype and analyzed using modified fixation indices (Weir and

Cockerham, 1984; Excoffier et al. 1992), and analysis of molecular variance (AMOVA;

Excoffier et al. 1992). The level of relatedness was significantly higher for haplotypes of the same voltinism ecotype at Lamberton and Rosemount, MN collection sites (é^T = 0.030; P =

0.008; Table 3). This suggested inbreeding within and reduced gene flow between voltinism ectypes. Furthermore, only 1.53% of total genetic variance between haplotypes was due to voltinism, again suggesting presence of low but significant levels of differentiation.

Sympatric voltinism ecotype differentiation was also detected via a pairwise comparison of genetic distance estimates (Table 4). Specifically, the sympatric uni- and 71

bivoltine ecotypes from Lamberton, MN showed a significantly different pairwise genetic

distance (D = 0.0534, P < 0.0001; Table 4), and may represent a measure of ecotype

variation without confounding geographic effects. In contrast, no significant genetic distance

was observed between uni- and bivoltine haplotype frequencies from Rosemount, MN (D =

0.0198; P = 0.054; data not shown). Genetic differentiation between sympatric voltinism

ecotypes at Lamberton, MN suggests a possible mating barrier that may be attributable to

mating period asynchrony (Eckenrode et al. 1983; Roelofs et al. 1985). Recent northern

bivoltine movement into Minnesota might be responsible for voltinism differences.

Univoltine ecotypes migrated to Minnesota in the early 1940s (Chiang 1961) and reached

North Dakota in 1950 (Chiang 1972), and a second O. nubilalis migration entered southern

Minnesota in 1952; suggested to be a northern expansion of bivoltine moths (Chiang et al.

1965; McLeod 1978; Palmer et al. 1985). Bivoltine migration into univoltine areas and

minimal gene flow due to non-overlapping mating periods may have maintained ecotype

differences. Alternatively, genetic drift in smaller northern populations may cause yearly

fluctuation in haplotype frequencies that caused Rosemount, MN samples to be above

threshold when sampled, or Bonferroni-corrected significance thresholds are unrealistically

high.

Significant differences in genetic distance estimates, ^-statistics, and AMOVA suggest little interspecific gene flow. Genetic diversity observed between sympatric O. nubilalis voltinism types may reflect the effects of reproductive isolation or ecological adaptation (Showers 1979; Showers 1993). Ecotype differentiation not associated with change in plant host range appears rare except in pea aphid, gallflies, soapberry bug, and brown plant hopper (see Berlocher and Feder 2002 for references). The extent of O. 72 nubilalis ecotype (reproductive) isolation in regions of sympatry remains to be determined, but development of more polymorphic genetic markers and more detailed sampling might help address this question.

Conclusions

Low but significant geographic subdivision of North American O. nubilalis population may exist (Table 2 and 4). Exclusion of an O. nubilalis Haelll mitochondrial haplotype from Atlantic coast populations might indicate a genetic bottleneck among

European founders, rarity of Haelll among immigrants from midwestern region of the United

States, or a migrant barrier imparted by the Appalachians. Data also showed genetic differentiation between sympatric O. nubilalis voltinism ecotypes, which may be maintained by asynchrony of mating periods and a reduced level of intermating (McLeod et al. 1979;

Eckenrode et al. 1983), or artifact of recent northern expansion of bivoltine ecotypes.

Additional genetic markers and more detailed sampling are required to further investigate these observations and currently are under development.

Acknowledgments

This research was a joint contribution from the USD A Agricultural Research Service and the

Iowa Agriculture and Home Economics Experiment Station, Ames, IA (project 3543). This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation by USDA or Iowa State University for its use. Thank you to all contributors of biological material from collection sites. 73

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Weir BS, Cockerham CC (1984) Estimating ^-statistics for the analysis of population structure. Evolution 38:1358-1270. 77

Figure 3.1. Location of 15 North American O. nubilalis collection sites. Ecotypes obtained from each collection site are indicated; univoltine Z pheromone race (UZ), bivoltine

Z pheromone race (BZ), univoltine E pheromone race (UE), or bivoltine E pheromone race

(BE). The New York (NY) laboratory colony data (site 3; lab) was omitted from statistical analysis due to haplotype fixation. Collection site ID: 1 Oxford Maine (ME); 2 Cape

Elizabeth, ME, 3 Ithaca, New York (NY); 4 Newark, Delaware (DE); 5 Columbia City,

Indiana (IN); 6 Franklin County, IN; 7 Crawfordsville, Iowa (IA); 8. Hubbard, IA; 9

Kanawha, IA; 10 Mead, Nebraska (NE); 11 Garden City, Kansas (KS); 12 Lamberton,

Minnesota (MN); 13 Rosemount, MN; 14 Brookings, South Dakota (SD); 15 South Shore,

SD.

KBE/UE)

15(u 2CBE/BZ) 14(u 13(BZ/UZ) 12(BZAfZ) 3(lab)l

•11 ( 78

Figure 3.2. Parsimony haplotype tree constructed from a 2,156 bp mitochondrial DNA alignment of 14 mitochondrial coxl, tRNA-LeuUUR, and cox2 DNA sequence (Table 1) randomly selected from the 1,414 O. nubilalis samples evaluated.

AF442957

-Cape Elizabeth, ME (UE) $46

- Oxford, ME (UE) $06

1000 -Cape Elizabeth, ME (BE)$47 160> Crawfordsville, IA $46

530 FranklinCo., IN $04 490I GardenCity, KS$17

Kanawha, IA $10

510I

140 Kanawha, IA $46 20 Lamberton, MN (BZ) ^24 190 10 Mead, NE S02

•Rosemount, MN (UZ)$20

10 Lamberton, MN (UZ)$26

Brookings, SD$06 270I Hubbard, IA c?25 79

Table. 3.1. Twenty-six variable nucleotide positions observed between 14 North

American O. nubilalis mitochondrial coxl and cox2 sequences. Random collection of sequences used to survey level and location of polymorphism among samples, such that

PCR-RFLP assays could be developed for higher sample throughput. Positions with respect to the O. nubilalis mitochondrial genome sequence (GenBank AF442957), and - indicates sequence identical to the consensus sequence. RFLPs: D = DdeI, H = Hae III, S = Sau3Al, and M = Mspl are indicated. See Table 2 for location detail of collection site ID.

coxl cox2 Position 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 4 5 0 0 1 1 1 1 5 5 5 8 8 9 9 0 1 4 4 4 4 4 5 5 5 5 Sequence 5 2 4 6 1 4 6 6 3 3 5 2 6 2 3 5 2 2 5 6 7 7 2 4 5 5 Site ID / 9 9 7 2 9 9 1 9 3 3 4 9 6 8 3 0 3 9 3 2 4 7 8 4 4 5 individual Consensus C T T T T A T A A T A T T T A G A A A A A A A A T A AF442957 A C C A - T G - T - G G - G - C ------G - 11/Ç17 ------G - G - T T T G T - - T 8/(325 - C G - - - - C - C - - G - - 14/$06 - - A ------T - G - - 12/^24 - - G - - - - c ------10/J02 G 9/Ç10 T 9/Ç46 - - - - T - c - - " - - - - - 12/$26 - c 13/$20 G 1/Ç06 - G 2/Ç47 C - - - 6/Ç04 ------T - - G - T 7/Ç46 T 2/G46 G - AAA I-M G-V I-M w-s I-M W-C G-V RFLP D H M S 80

Table 3.2. Distribution of mitochondrial RFLP frequencies and haplotypes in samples

from 15 North American collection sites. Frequency of digestion (PCR-RFLP frequency),

and number of O. nubilalis with a given haplotype at each collection site (mitochondrial

haplotype number) are provided. Note: Maine (ME) and Minnesota (MN) samples were

divided into ecotype (ET) for genetic distance estimation (see Table 4).

PCR-RFLP frequencies Mitochondrial haplotype numbers Subpopulation ET D H S M w+ D H s M DM DS MS DH DH ID (collection site) M 1 Oxford, ME U UE 100.00 0.00 10.41 0.00 43 0 0 5 0 0 0 0 0 0 UZ 100.00 0.00 18.75 0.00 13 0 0 3 0 0 0 0 0 0 2 Cape Elizabeth, BE 100.00 0.00 6.25 0.00 90 0 0 6 0 0 0 0 0 0 ME U BZ 100.00 0.00 6.25 4.17 43 0 0 3 2 0 0 0 0 0 3 Ithaca NY Y BE 100.00 0.00 0.00 0.00 48 0 0 0 0 0 0 0 0 0 (lab colony) BZ 100.00 0.00 0.00 0.00 48 0 0 0 0 0 0 0 0 0 4 Newark, DE R BE 97.92 2.08 2.08 2.08 46 0 0 1 0 0 0 0 0 1 5 ColombiaCity,INS BZ 97.96 3.06 2.04 2.04 90 1 2 2 2 0 0 0 1 0 6 FranklinCo, IN S BZ 100.00 2.35 1.18 3.53 79 0 2 1 3 0 0 0 0 0 7 Crawford ville,IA) BZ 96.88 6.25 2.08 0.00 85 3 6 2 0 0 0 0 0 0 8 Hubbard, IA BZ 100.00 7.29 2.08 2.08 86 0 7 1 1 0 0 1 0 0 9 Kanawa, IA BZ 100.00 2.35 1.18 3.53 79 0 2 1 3 0 0 0 0 0 10 Mead, NE X BZ 98.96 8.33 3.13 1.04 82 1 8 3 1 0 1 0 0 0 11 GardenCity,KST MZ 96.47 4.71 4.71 5.88 81 2 4 4 4 1 0 0 0 0 12 Lamberton, MNV BZ 96.83 0.00 4.69 4.69 56 2 0 3 3 0 0 0 0 0 UZ 96.43 10.71 0.00 0.00 40 2 6 0 0 0 0 0 0 0 13 Rosemount,MNV BZ 100.00 6.45 4.30 0.00 83 0 6 4 0 0 0 0 0 0 UZ 87.50 18.75 9.38 9.38 32 0 6 2 2 0 0 1 0 0 14 Brookings, SD Z UZ 94.44 1.85 3.70 3.70 46 3 1 2 2 0 0 0 0 0 15 South Shore,SDZ UZ 100.00 3.74 0.00 5.61 97 0 4 0 6 0 1 0 0 0 Total 98.66 3.96 3.47 2.19 1267 14 54 45 27 1 2 2 1 1

Ecotypes (ET) represented are univoltine Z pheromone race (UZ), bivoltine Z pheromone race (BZ), univoltine E pheromone race (UE), or bivoltine E pheromone race (BE). Haplotypes defined usingD = Ddel, H = Haelll, S = Sau3 AI, M = Mspi, and w+ (wildtype) uncleaved. Collections provided by: R. Charles Mason., 2001 U. David Handley 2002 X. Ron Seymour 2001 S. John Obermeyer 2001 V. Bruce Potter 2001 & 2002 Y. Wendel Roelofs 2001 T. Lawrence Buschman 2001 W. William Hutchison 2002 Z. Micheal Catangui 2001 81

Table 3.3. Mitochondrial AMOVA and modified ^-statistics (Theta (^-statistics; Weir and Cockerham, 1984). A) Subdivision of North American haplotypes into Atlantic coast

(sites 1, 2, and 5) and midwestern collection sites (sites 5-15, Table 2; Ithaca, NY laboratory colony haplotype data was omitted). B) Subdivision of sympatric univoltine (UZ) and bivoltine (BZ) ecotypes from Lamberton and Rosemount, MN collection sites (sites 12 and

13; Table 2).

A) Atlantic and midwestern subdivision df Sum of Variance % of squares component a2 Among regions 1 0.946 0.00188 Va 1.64 Among subpops 14 2.757 0.00082 Vb 0.71 within regions Within subpops 1300 146.141 0.11242 Vc 97.65 Total 1317 149.844 0.11512 100.00

Fixation Indices (6) f-value and SE Bsc (Fis) 0.00725 0.00098 ± 0.00098

6st (FST) 0.02350 0.00000 ± 0.00000 9ct (Fn) 0-01637 0.00098 ± 0.00098 B) Minnesota sympatric voltinism df Sum of Variance %of squares component a2 Among ecotypes 1 0.529 0.00227 Va 1.53 Among subpops 2 0.544 0.00212 Vb 1.43 within groups Within subpops 244 35.008 0.14348 Vc 97.03 Total 1413 36.081 0.14786 100.00

Fixation Indices (0) P-value and SE Bsc (F,s) 0.01455 0.30108 ±0.01070 Bst (Fst) 0.02966 0.00782 ±0.00313 6ct (F1T) 0.01533 0.33138 ±0.00000 82

Table 3.4. Significantly different pairwise Nei genetic distance (D) comparison among mitochondrial haplotypes from an 18 x 18 matrix of 14 O. nubilalis sample sites (Ithica,

NY laboratory colony data omitted). Empirical P values were determined after 10,000

Markov chain steps (permutations), and significance threshold was set using the Bonferroni adjustment for multiple tests (0.05/153 = 0.0003267). Note: Haplotypes from Lamberton and

Rosemount, MN, and Cape Elizabeth and Oxford, ME collection sites subdivided into ecotype (Table 2). See Table 2 for collection site ID and ecotype (ET) definition.

ID Collection site ET ID Collection site ET D P-value 1 Oxford, ME UE vs. 6 Franklin Co., IN BZ 0.0470 <0.0001 10 Mead, NE BZ 0.0869 <0.0001 12 Lamberton, MN UZ 0.0929 <0.0001 13 Rosemount, MN UZ 0.0785 <0.0001 15 South Shore, SD UZ 0.0326 <0.0001 Oxford, ME UZ 5 Columbia City, IN BZ 0.1005 <0.0001 12 Lamberton, MN UZ 0.0654 <0.0001 2 Cape Elizabeth, BE vs. 7 Crawfordsville, IA BZ 0.0278 <0.0001 8 Hubbard, IA BZ 0.0328 <0.0001 9 Kanawha, IA BZ 0.0225 <0.0001 12 Lamberton, MN UZ vs. 6 Franklin Co., IN BZ 0.0487 <0.0001 9 Kanawha, IA BZ 0.0487 <0.0001 2 Cape Elizabeth BE 0.0851 <0.0001 12 Lamberton, MN BZ 0.0534 <0.0001 13 Rosemount, MN UZ vs. 5 Columbia City, IN BZ 0.0424 <0.0001 9 Kanawha, IA BZ 0.0484 <0.0001 83

CHAPTER 4. POLYMORPHIC CA/GT AND GA/CT MICROSATELLITE LOCI FOR OSTRINIA NUBILALIS (LEPIDOPTERA: CRAMBIDAE)

Brad S. Coates, Douglas V. Sumerford, Richard L. Hellmich, and Leslie C. Lewis

Paper published: Molecular Ecology Notes, 5(1):10—12, 2005

Abstract

Ten polymorphic dinucleotide (CA/GT and GA/CT) microsatellite loci suitable for population genetic screening were characterized from enriched partial Ostrinia nubilalis genomic libraries. Sequence from 126 enriched small insert genomic library clones identified 25 CA/GT and 58 GA/CT loci that were unique. Perfect repeats tended to be short

(n = 10 to 12). Ten microsatellites PCR amplified from a Crawfordsville Iowa population showed a mean of 10 alleles per locus (range 6 to 20), and 6 of 10 loci showed hétérozygote deficiency. Amplification of 8 loci was observed in the sister species O.furnicalis.

Keywords: Lepidoptera, Ostrinia nubilalis, genetic marker

Short Note

Microsatellite markers are highly polymorphic and codominant, and useful for population genetic and genome mapping studies (Goldstein and Schlotterer 1999). Microsatellite isolation from Lepidoptera has been difficult (Nève and Meglècz 2000) and few reported from moths (Reddy et al. 1999; Ji et al. 2003).

Larval European corn borer, Ostrinia nubilalis (Hubner; Lepidoptera: Crambidae) infest Zea mays and cause major yield loss (Showers 1993). Coates and Hellmich (2003) showed geographic variation among O. nubilalis using two sex-linked markers, but 84 acknowledged need from additional markers. Ostrinia nubilalis history and biology is of interest. First, O. nubilalis is native to Europe and western Asia, and genetic bottleneck and range expansion since North American introduction suggests a model for species introduction. Secondly, sympatric ecotypes differing in pheromone use, and generation number (voltinism) exist (Showers 1993). Co-existence and maintenance of distinct ecotypes suggests potential population and ecological studies.

Two dinucleotide microsatellite enriched partial O. nubilalis genomic libraries

(CA/GT and GA/CT) were constructed from 12 individuals. Genomic DNA extracts were

prepared using DNAeasy kits (Qiagen) and 1.25 jag digested in 50 pi reactions containing

10U EcoRl (Promega), 10U Msel (New England BioLabs), 100 ng each Msel and EcoKl

adapter (Vos et al. 1995), 10U T4 DNA ligase (Promega), 5 |LX1 NEB buffer 2, and 5 pM ATP

incubated 14 hr at 37°C. Ligations (250 ng) were mixed with 100 pmol 5' biotin-TEG

(CA)i2 or (GA)i2 probes in 100 pi IX SSC, fragments denatured at 95°C for 5 min, and

hybridized at 55°C for 5 min on a PTC-100 thermocycler (MJ Research). Streptavidin

paramagnetic beads (100 (j.1; Promega) were added and incubated for 5 min, then washed

twice with 800 pi IX SSC at 55°C for 5 min. DNA was released by incubation with 100 pi

80°C deionized water for 5 min.

Recovered DNA fragments (10 pi) were PCR amplified in 50 pi reactions with 5 pi

10X thermal polymerase buffer (Promega), 1.5 mM MgCli, 200 pM dNTPs, 3.5U Taq polymerase (Promega), and 30 pmol £coRI adapter primer (5'-GAC TGC GTA CCA ATT

C-3') and Msel adapter primer (5'-CGA TGA GTC CCT GAG TAA-3'). A PTC-100

thermocycler used 30 cycles of 96°C for 20 sec, 52°C for 30 sec, and 72°C for 45 sec, 85 followed by 72°C for 20 min. PCR product (1 pi) was ligated into 50 pmol pGEM-T easy cloning vector (Promega). Plasmids were cloned into E. coli SURE (Stratagene) by electroporation, recombinants identified by blue/white screening, isolated by alkaline lysis, and purified by PEG precipitation (Sambrook et al. 1989). Ten microliter (10 pi) DTCS

Quickstart DNA sequencing reactions (Beckman-Coulter) used 1.6 pmol T7 primer.

Sequence reaction products were purified by ethanol precipitation, suspended in 40 pi deionized formamide, and separated on a CEQ 8000 Genetic Analysis System (Beckman-

Coulter) with method LFR-1 (denature: 90°C for 120 sec; inject: 2.0 kV for 15 sec; and separated: 4.2 kV for 85 min in a 50°C capillary).

Sequence from 126 plasmid inserts had 102 microsatellite loci, and 83 were unique;

25 CA/GT and 58 GA/CT microsatellites. Most repeats were short (n = 10-12bp). PCR primer pair design was attempted for 44 cloned sequences containing perfect repeats using

Primer3 (Rozen and Skaletsky 1998). Acceptable PCR primers were not obtained from 14 sequences due to low GC content (< 40%), low Tm (< 55°C), or self-annealing primer pairs.

From 30 synthesized primer pairs (Integrated DNA Technologies) 10 PCR products were polymorphic, had < 2 bands/individual, and were within predicted size ranges (Table 4.1).

Forward or reverse primer was synthesized and 5'-labeled with WellRed® dye (D2,

D3, or D4; Proligo) for the 10 acceptable loci. PCR reactions contained 1.5 mM MgClz

(loci: OnGA15, OnGA32, OnCA14, OnCA46, and OnCA41) or 2.5 mM MgCl2 (all remaining loci; Table 4.1), 150 pM dNTPs, 50 ng DNA, 2 pmol of each primer, 1 pi 10X thermal polymerase buffer (Promega), and 0.3125U Taq DNA polymerase (Promega). A

PTC-100 thermocycler used 96°C for 3 min, then 35 cycles of 96°C for 20 sec, 30 sec annealing (see Table 4.1), and 72°C for 30 sec. All touchdown (TD) PCR used stepdown from 67°C at -2 °C/cycle for 7 cycles, then 35 cycles with 52°C annealing; other parameters identical to standard PCR. PCR product (0.5 pi) was added to 40 pi deionized formamide and separated on a CEQ 8000 Genetic Analysis System (Beckman-Coulter) using method

Frag-3 (denature: 90°C for 120 sec; inject: 2.0 kV for 30 sec; and separated: 6.0 kV for 35 min in a 50°C capillary) with 0.25 pi 400 bp internal standard (Beckman-Coulter).

Allele number/locus ranged from 6-20 (mean 10 ±3.944) among 61-91 O. nubilalis collected near Crawfordsville, Iowa (Table 4.1). Expected heterozygosity (HE) was calculated according to Nei (1973) and differences with observed heterozygosity (Ho) tested by chi-square (%2). Calculations used POPGEN software v. 1.32 (Table 4.1; Yeh and Boyle

1999). Four of 10 O. nubilalis microsatellite loci were in Hardy-Weinberg equilibrium

(HWE) and remainder (60%) showed heterozygote deficiencies. High O. nubilalis homozygosity may be due to genetic bottleneck at North American introduction, range expansion, or local inbreeding (Hartl and Clark 1997). Arlequin software detected no linkage disequilibrium between loci (results not shown; Schneider et al. 1997).

Acknowledgments

Mention of proprietary products does not constitute an endorsement or a recommendation by

USDA or Iowa State University for its use. 87

References

Coates BS, Hellmich RL (2003) Two sex-linked microsatellite loci show geographic variance among North American Ostrinia nubilalis. J Insect Sci 2:29.

Goldstein DB, Schlotterer C (1999) Microsatellites: evolution and applications. Oxford University Press, Oxford, U.K.

Hartl DL, Clark AG (1997) Principles of Population Genetics. Sinauer, Sunderland, MA.

Ji YJ, Zheng DX, Hewitt GM, Kang L, Li DM (2003) Polymorphic microsatellite loci for the cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) and some remarks on their isolation. Mol Ecol Notes 3:102-104.

Nei M (1973) Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89:583-590.

Nève G, Meglècz M (2000) Microsatellite frequencies in different taxa. Trend Ecol Evol 15:376-377.

Reddy KD, Abraham EG, Nagaraju J (1999) Microsatellite in the silkworm, Bombyx mori: abundance, polymorphism, and strain characterization. Genome 42:1057-1065.

Rozen S, Skaletsky HJ (1998) Primer3. Available from http://genome.wi.mit.edu/genome_software/other/primer3.html.

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: a Laboratory Manual 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Schneider S, Kueffer JM, Roessli D, Excoffier L (1997) Arlequin ver.1.1: a software for population genetic data analysis, http://lgb.unige.ch/arlequin/

Showers, WB (1993) Diversity and variation of European corn borer populations, pp. 287- 309. In Kim KC, McPherson BA [eds.] Evolution of Insect Pests/Pattems of Variation. Wiley and Sons Inc., New York, NY.

Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Homes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP: a new technique for DNA fingerprinting. Nuc Acids Res 23:4407^1414.

Yeh CF, Boyle T (1999) POPGEN VERSION 1.32: Microsoft Window-based freeware for population genetic analysis, http://www.ualberta.ca/~fyeh/ Table 4.1. Ten Ostrinia nubilalis CA/GT and GA/CT microsatellite markers. Primers, repeat unit, GenBank accession, annealing temperature (Tm), PCR product size range, allele number, and sample number are given for each locus. The 5'-labeled primers are indicated with appropriate WellRed® dye (D2, D3, or D4; Proligo). The observed heterozygosity (H0) was obtained from a Crawfordsville, Iowa population sample and expected heterozygosity (HE) calculated according to Nei (1973). # indicates significant difference between HE and HQ. Ostrinia furnicalis (O.furn.) amplification success using O. nubilalis PCR reaction parameters (see methods) was judged from 4 samples.

GenBank Tm PCR size Allele O.f. Locus Primer sequence Repeat accession (°C) range (bp) No. N. H0 He +/— OnGA16 F-D2-CTGCGTACATGCAGCGTAAA (GA)n AY642964 TD 124-134 6 75 0.393 0.643 # + R-TCATCACCATCGATGAGTCC OnGA28 F-D4-CTGCGTACATGCAGAATAAGGG (GA)., AY642965 52 86-104 8 87 0.368 0.742 # + R-TTTTATCAAACTCTTAYRCGATGAAGC OnGA32 F-D4-GGCCTGATCATTTCCCTGAG (CT),S AY642967 55 150-166 8 89 0.461 0.487 + R-TGCTGCATGTACGCAGTCAG OnGA33 F-D3-TCAAACTCTTATGCGATGAAGC (GA)u AY642968 59 73-105 13 91 0.528 0.637 + R-CTGCAGAATAAGGGGCACT OnGA39 F-D3-TCTCAAGCACTTTGATCCGG (CT)« AY642969 TD 88-104 9 86 0.198 0.606 # + R-GGCTGAAGTTGGAAGAGGATCT

OnCAO? F-D2-GAATTCATTTTACTCAAGATTTGTTTA (CA)u AY642970 55 115-133 8 67 0.210 0.755 # — R-TAATATGGCCTGATGGTGTGCA OnCA16 F- D4-CCTGACTTGTGCCGAGTAGGT (CA), AY642971 TD 127-145 10 61 0.613 0.727 + R-AACATGGCCGCAATATAGGC OnCA27 F-D2-CCTGACTTGTGCCGAGTAGGT (CA)1S AY642972 TD 126-146 9 89 0.562 0.626 + R-C A ACAT GGCCGCAAT ATAGGC OnCA41 F-D2-GCCTAAGGCAGGAATTGAACC (CA)„ AY642973 TD 189-247 20 86 0.439 0.855 # R-CGGAAACCTTACTCCAAACTCTC OnCA46 F-D2-GTAGGGCTACCCATCACGCACA (CA)IO AY642974 57 67- 83 9 88 0.455 0.733 # + R-C A A A AGT A AGGT AAGGCT AT GT G AC 89

CHAPTER 5. TWO SEX-CHROMOSOME-LINKED MICROS ATELLITE LOCI SHOW GEOGRAPHIC VARIANCE AMONG NORTH AMERICAN

Brad S. Coates and Richard L. Hellmich

Paper published: International Journal of Insect Science, 3:29, 2003

Abstract

PCR-based O. nubilalis population and pedigree analysis indicated female specificity of a

(GAAAAT)n microsatellite, and male specificity of a CAYCARCGTCACTAA repeat unit marker loci respectively were named Ostrinia nubilalis W-chromosome 1 (ONW1) and O. nubilalis Z-chromosome 1 (ONZ1). Intact repeats of three, four, or five GAAAAT units are present among ONW1 alleles, and biallelic variation exists at the ONZ1 locus. Screening of

493 male at ONZ1 and 448 heterogametic females at ONZ1 and ONW1 loci from eleven

North American sample sites was used to construct genotypic data. Analysis of molecular variance (AMOVA) and /^-statistics indicated no female haplotype or male ONZ1 allele frequency differentiation between voltinism ecotypes. Four subpopulations from northern latitudes, Minnesota and South Dakota, showed absence of a single female haplotype, significant deviation of ONZ1 data from Hardy-Weinberg expectation, and low-level geographic divergence from other subpopulations. Low ONZ1 and ONW1 allele diversity was attributed to large repeat unit sizes, low repeat number, reduced effective population

(Ne) size of sex chromosomes, or result of recent O. nubilalis introduction and population expansion, but not inbreeding.

Key Words: Ostrinia, Pyraloidea, microsatellite, sex-linked genetic markers. 90

Abbreviations PCR Polymerase chain reaction ONW1 Ostrinia nubilalis W-chromosome marker number 1 ONZ1 Ostrinia nubilalis Z-chromosome marker number 1

Introduction

Microsatellites are repetitive nucleotide elements that have a core repeat structure of 2 to 6 nucleotides (Chambers and MacAvoy 2000). Microsatellite allele mutations predominantly occur via strand slippage during chromosome replication at a rate of 10"6 to

10~2 (Eisen, 1999), and influenced by repeat unit length, number of repeat units in the array, microsatellite flanking sequence, and recombination (Chambers and MacAvoy 2000).

Ostrinia nubilalis (Hiibner), the European corn borer, is an invasive agricultural pest in North America introduced from Europe around 1917 and herbivorous O. nubilalis larvae adapted to feeding on cultivated Zea mays (L.) annually causing major economic loss (Mason et al. 1996). Phenotypic diversity in the North American population is present in two sex pheromone races, and three voltinism types (Showers 1993). Females of the two sex pheromone races are differentiated by their emission of either E- or Z-11-tetradecenyl acetate as the dominant pheromone component. Pheromone races show little allozyme marker differentiation (Harrison and Vawter 1977; Cardé et al. 1978; Glover et al. 1991), and interbreed in laboratory and natural conditions suggest continual gene flow (Roelofs et al.

1985; DuRant et al. 1995). In contrast, diapause ecotypes differ in the number of degree days required prior to adult emergence (Showers et al. 1993), and resultant asynchrony of adult mating cycles between voltinism types was suggested to minimize genetic exchange

(Roelofs et al. 1985). Reduced gene flow between voltinism ecotypes was supported by 91

RAPD-PCR marker data (Pomkulwat et al. 1998), but not by allozyme marker (Bourguet et

al. 2000) or mitochondrial RFLP data (Marcon et al. 1999).

To date, locus specific genetic markers used for O. nubilalis population analysis have

inherent limitations that restrict levels of variation detected, which might contribute to the

conflicting evidence provided between O. nubilalis ecotypes. Intraspecific diversity of

allozyme markers is reduced by negative selection on nonsynonymous changes and

environmental influence on posttranslational modification pathways (Hartel 1988), and

mitochondrial DNA is prone to fixation by genetic drift due to a reduced effective population

size of the molecule compared to chromosomes (Avise et al. 1988). Given limitations of

previously used markers, unresolved questions regarding gene flow among North American

ecotypes, and desire to estimate both male- and female-based population differentiation

(movement) we developed sex-specific O. nubilalis nuclear microsatellite markers. In the

following we characterize allele variation of the North American population at two such markers, ONW1 (Ostrinia nubilalis W-chromosome 1) and ONZ1 (Ostrinia nubilalis Z- chromosome 1). Genotypic and haplotypic data were used evaluate intraspecies differences, infer presence of any population subdivision, and estimate levels of genetic exchange

(migration) among O. nubilalis voltinism ecotypes and geographically distinct North

American subpopulations.

Materials and Methods

Satellite DNA Isolation

Genomic DNA from O. nubilalis was enriched for CA microsatellites by methods described by Lyall et al. (1993). Resultant PCR products were ligated into pGEM-T easy cloning 92 vector (Promega, Madison, WI) according to manufacturer instructions. Electroporation competent E. coli SURE® cells (Stratagene, La Jolla, CA) were transformed on a MicroPulsar appratus (BioRad, Hercules, CA), and clone selection and blue white screening was performed.

Positive clones were propagated overnight at 37°C in 25 ml Terrific Broth that contained ampicillin. Plasmid DNA was isolated with a QIAprep spin miniprep kit (Qiagen, Valencia, CA) according to manufacturer's directions. Template was sequenced at the DNA Sequencing and

Synthesis Facility at Iowa State University, Ames, IA, USA.

Marker Screening and Analysis

Ostrinia nubilalis samples were collected from three light traps in Iowa, and samples obtained from collaborators (Table 5.1). DNA extractions were performed on moth thoracic tissue as described by Marcon et al. (1999). DNA pellets were diluted to 50ng/pl with TLE

(10 mM Tris, 0.1 mM EDTA, pH 7.5) and stored at - 20 °C prior to use. Primer pairs

ONW1-F (5'- TGGAAGTT GAT C GGAAT AAGAAGT C -3') with ONW1-R (5'-

TGGAAGAGCGGTAACCTCCT -3'), and ONZ-M1F (5'-GGTGGGACCTCCATGCGC-

3') with ONZ-M1R (5'-GCTGGGGCGTCTTCGAGGT-3') were designed from respective

DNA sequence data using Primer3 (Rozen and Skaletsky 1998). Primers were synthesized at

Integrated DNA Technologies (Coralville, IA), and 5 pmol of each used along with 0.425 U

Taq polymerase (Promega), 1.25 pi 10X thermal polymerase buffer (Promega), 2.5 mM

MgClz, and 150 pM dNTPs to PCR amplify 150 ng of individual O. nubilalis DNA sample in a 12.5 pi reaction volume. Thermocycler reactions used 94°C for 3 min., followed by 40 cycles of 94°C for 20 sec., 53°C for 30 sec. (ONW1) or 50°C for 30 sec. (ONZ1), and 72°C 93 for 15 sec. ONW1 locus amplification from eighteen other species used modified amplification conditions (Table 5.2). PCR products were separated at 150V for 10.5 hr. on a

0.1 x 20 cm 8% polyacrylamide (19:1 acrylamide:bisacrylamide) IX TBE gel with a 25 bp ladder (Promega) for size comparison. Bands were visualized after ethidium bromide staining, and image capture took place on a PC-FOTO/ Eclipse Electronic Documentation

System (Fotodyne, Hartland, WI). Basis of variation was determined by cloning > 2 loci of each observed fragment size for ONW1 and ONZl. Plasmid DNA isolation and DNA sequencing was carried out as previously described.

ONW1 allele and ONZl genotypic frequency data was used to evaluate O. nubilalis population structure, and estimate male and female migration (M) between subpopulations.

Population structure assumed genetic separation of univoltine Z-pheromone subpopulations

(UZ; ID 1 to 4), from bivoltine and multivoltine Z-pheromone (Table 5.1; ID 5 to 11).

Calculations of geographic based on used three groups: group 1, subpopulations with ID 1 to

4; group 2, ID 5 to 9; group 3, ID 10 and 11. A second analysis assumed north-south population subdivision assumed two subpopulation groups; north: subpopulations with ID 1 thru 4, and south ID 5 to 11 (Table 5.1). Statistical evaluations included analysis of molecular variance (AMOVA), and ^-statistic or haploid allele data modified F-statistic (#

Excoffier et al. 1992; Weir and Cockerham 1983; Weir 1996). Subpopulation ONZl Hardy-

Weinberg equilibrium (HWE) was tested using 10,000 Markov chain steps, and significance p values set at 0.05. All calculations were performed with the Arlequin software package

(Schnieder et al. 1997). 94

Results

Two polymorphic tandem repetitive loci O. nubilalis Z-chromosome 1 (ONZl) and

Ostrinia nubilalis W-chromosome 1 (ONW1) were sequenced from separate clones in a microsatellite enriched partial genomic library. The ONW1-containing clone, pGEM-

QnCA09, had two repetitive regions, ten tandem GT/CA repeat units and four consecutive

GAAAAT repeats (Fig. 5.1a; GenBank AF442958), and population analysis revealed three alleles of 141 (3 repeats; allele WA3), 147 (4 repeats; allele WA4), and 153 bp (5 repeats; allele WA5). Two samples from each of three observed ONW1 allelic types (electromorphs) were DNA sequenced, and full GAAAAT repeat units identified as basis of size variation without flanking region mutation (data not shown). The ONW1 hexanucleotide repeat showed successful PCR from only female DNA samples (data not shown). The ONZ1- containing clone pGEM-OnCAOl had a fifteen nucleotide-long imperfect repeat element,

CAYCARCGTCACTAA, (underlined nucleotides optional; Fig lb; GenBank AY102618 to

AY 102620). ONZl was PCR amplified from male derived DNA and all attempts to amplify

female DNA failed (data not shown). Six ONZl alleles were sequences, three from each of two size classes, two ONZl allele size range variants 95 to 97 (ZAt) and 106 to 107 bp (ZA2) were characterized with one insertion/deletion and two substitutions within both the repeat

and the flanking region (Fig lb). Since single base changes were not resolved by

polyacrylamide gel electrophoresis methods, two alleles (ZAj and ZA2) were scored and used

for genotype data. Three pedigrees with > 46 F2 progeny confirmed male specificity of

ONZl and female specificity of ONW1. More specifically, ONW1 and ONZl were hemizygous in females (single locus with no corresponding homologous pair), and males were diploid at ONZl and lacked the ONW1 locus. 95

ONZl and ONW1 were PCR amplified from 448 female, and ONZl from 493 male

O. nubilalis DNA samples from 11 North American subpopulations. Male ONZl genotypic

and ONZl and female O. nubilalis ONW1 haplotype frequencies were calculated (Table

5.1). Ostrinia nubilalis male genotypic and female haplotype data were separately evaluated

by AMOVA and fixation indices, and independently showed stronger evidence for

geographic- compared to voltinism-based population structuring (Table 5.2).

Discussion

Low allele size diversity was present at O. nubilalis W chromosome (ONW1) and O.

nubilalis Z-chromosome (ONZl) microsatellite loci. The ONW1 allele with four repeat units

[(GAAAAT)4; WA4), was present among 76% of North American O. nubilalis females, and

described from 87.1 % of 41 female O.furnicalis (Gunée) and a single O. pentitalis (Grote)

sample, indicating the allele likely is ancestral to the genus. Molecular basis of low ONW1 allele

size variation might lie in repetition of a hexanucleotide repeat that is less prone to strand slippage

during recombination, the short array of 3 to 5 GAAAAT repeats, and potential location of ONW1

in the nonrecombining region of the W chromosome that eliminates unequal crossover as a

mechanism generating allele diversity. ONW1 WA3 alleles outnumbered WA5 alleles, 19.3% to

4.7% (Table 5.1), indicating a preponderance of microsatellite repeat unit loss, as opposed to gain that occurs typically at microsatellite loci (Rubinsztein et al. 1995; Weber and Wong 1995). Rose and Falush (1998) determined a minimum of eight repeats was required for evolution of hypervariable microsatellites, and array lengths such as ONW1 that are below this threshold suggest insensitivity toward repeat number expansion bias. The ONZl locus is biallelic, with the dominant ZAi allele frequency at 95% (937 of 986) in males and 99% (445 of 448) in females. 96

Near fixation of ZA\ in female 0. nubilalis and not in males might result from differences in effective population sizes (Ne) of the W chromosome compared to Z chromosomes. Assuming a

1:1 sex-ratio, lepidopteran W chromosomes respectively have a three- and fourfold reduction in effective population size compared to Z and autosomal chromosomes (Charlesworth et al. 1987;

Hartel 1988), whereby associated loci are be more prone to fixation and interpopulation divergence

(Charlesworth et al. 1987). Despite being less prone to molecular fixation, ONZl locus allele diversity was lower than ONW1 that might result from its longer imperfect repeat, or be a result of population effects (discussed later).

No genetic divergence was detected between female ONW1 and ONZ haplotypes based on voltinism ecotype, as indicated by 6.44% (0.0%) of total population variation among groups.

Similarly at the ONZl locus, grouping samples by voltinism showed an FST = 0.028, and 0.83% of total population variance among ecotypes (remaining data not shown). Results indicated no evidence of O. nubilalis voltinism-based ecotype structure and are in agreement with mitochondrial PCR-RFLP (Marcon et al. 1999) and allozyme data (Bourget et al. 2000), but in conflict with voltinism ecotype variation detected by RAPD-PCR (Pornkulwat et al. 1998).

Additionally, a single S-pheromone subpopulation from Newark, DE did not show significant

ONZl allele or genotype variance with Z-pheromone race subpopulations. A trio se phosphate isomerase (TPI) marker previously was mapped to the Z chromosome and linked to inter- pheromone race behavioral or response differences in males (Glover et al. 1991), suggesting a Z chromosome location of ONW1 distant from TPI and male pheromone response genes. Lack of

both voltinism ecotype-based population differentiation or correlation of ONZl with pheromone

race might be an artifact of recent O. nubilalis range expansion across North America (Showers 97

1993), suggesting slowed ONW1 and ONZl lineage sorting (Avise et al. 1984) and allele

extinction by genetic drift (Takahata 1983) between ecotypes.

Low but significant differentiation previously was shown between O. nubilalis ecotypes

(Harrison and Vawter 1977; Cardé et al. 1978), but not between geographically distant samples

(Bourget et al. 2000). Both sex-linked genetic markers, ONW1 and ONZl, indicated greater

contribution of geography to total genetic variance compared to voltinism. AMOVA analysis indicated that 3.78% of total population variance was between two groups; a "northern" group consisting of Minnesota and South Dakota subpopulations (ID 1 to 4), and all other subpopulations classified as "southern" (ID 5 to 11; Table 5.1). Combined ONZl and ONW1 female haplotypes data indicated 4.02% of total population variance was accounted for among "northern" and

"southern" groups. Low-level geographic divergence of four subpopulations from northern latitudes might reside in their omission of the ZA1WA5 female haplotype (Table 5.1), and significant deviation of ONZl data from Hardy-Weinberg expectation at three of the four sites. In the "northern" geographic region, chi-square (%2) tests detected significant departure of ONZl genotypes from HWE within Lamberton, MN bivoltine Z-pheromone (p < 0.001), Lamberton, MN univoltine Z-pheromone (p = 0.007), and South Shore, SD univoltine Z-pheromone subpopulations

(p = 0.016). Deviation for HWE was not exhibited from any other subpopulation. Except for the

Brookings, SD sample, the basis for Hardy-Weinberg deviations among northern subpopulations likely reside in their decreased the average heterozygosity (H$ = 2^- 81 = 0.025) compared to the average among the remaining subpopulations (Hj =19-^317 = 0.060). Reduced heterozygosity might be attributed to presence of null alleles, or stronger effect of the % reduction in Z chromosome effective population size in smaller fringe populations. Alternatively, recent or recurrent population size effects might disproportionately disrupt O. nubilalis population HWE in 98 northern regions. Increased severity of cyclical genetic bottlenecks due to larval over-wintering mortality in northern regions (Hudson and LeRoux 1986) might disturb mutation-drift equilibrium since Ne likely does not remain stable for 2Ne to AN,, generations (Nei and Li 1976). Additionally, entry of univoltine O. nubilalis moths into Minnesota in the early 1940s (Chiang 1961), South

Dakota in 1948, and North Dakota in 1950 (Chiang 1972), followed by northern migration of bivoltine subpopulations starting in the early 1950s (Chiang 1965), suggest recent range expansion might have caused a deviation from migration-drift equilibrium (Takahata 1983).

Level of migration was inferred from ^-statistics derived from male genotype and female haplotype data (Table 5.2). Female haplotype analysis indicated substantial effects of inbreeding and genetic drift (6^T = 0.227; % = 0.274), whereas male ONZl genotypic analysis suggested little evidence of either (F$T = 0.028; Fis = 0.020; Table 5.2). Inbreeding prevalence among female O. nubilalis might be used to infer lower migration rates as compared to males. When considered in conjunction with increased levels of genetic drift among females (6^t = 0.227) in relation to males (Fis = 0.020), reduced effective population size of the W chromosome and lack of homologous recombination during female gametogenesis might result in a strong influence of nucleotide fixation in female lineages. Thus differences between loci used in this study might reflect a background of chromosome position effects, and not truly be representative of population- based influences. Corroborating biological estimations of male and female flight distances are required to verify our results.

Low ONZl and ONW1 allele diversity was attributed to large repeat unit sizes, low repeat number, reduced effective population (Ne) size of sex chromosomes, or result a genetic bottleneck at O. nubilalis introduction or genetic drift caused by continued population expansion. Analysis of male ONZl genotypes and female ONW1 and ONZl haplotypes showed no evidence for 99 voltinism-based population structure. The similarity of west-central Minnesota and eastern South

Dakota samples, compared to all other subpopulations, for both ONW1 and ONZl provided evidence of geographic population structure. Recent O. nubilalis population expansion into northern regions of the central United States and recurrent seasonal bottlenecks likely explain their maintenance of HW disequilibrium, and form the basis of geographic population subdivision instead of inbreeding.

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Figure 5.1. Sex linked microsatellite sequences. A) 272 bp insert DNA sequence from clone pGEM-OnCAQ9 showing three tandem GAAAAT repeats (underlined) and primer binding sites (underscored by arrows indicating direction) used to PCR amplify the locus

Ostrinia nubilalis W-chromosome marker number 1 (ONW1 ; GenBank accession:

AF442958). B) Multiple DNA sequence alignment of the PCR amplified O. nubilalis Z- chromosome linked marker number 1 (ONZl) alleles. Consensus sequence of imperfect alleles, CAYC ARC GT C ACT AA (underlined nucleotides optional), and primer binding sites

(arrows indicating direction) are underscored, and - representing an invariable nucleotide identical to clone OnCAOl, and * is a deletion.

A: nt 0 01 G AACAGGTAA GTTTGCGTGG TATACGAAGA CGAGTAAGAT TTGGAAGTTG ATCGGAATAA ONW1-F >>>>>>>>> >>>>>>>>>> 061 GAAGTCCATG AAAATGAAAA TGAAAATGAA AATGTTTTAT TTGATAAATA AAGTGGATTA

121 GCAAGATATT TTGGTAACCA TCCTTTTAAG TATAAGAATA CCTGTAGTAG GAGGTTACCG << <<<<<<<<<< 181 CTCTTCCAAA AGTATTAACA TACATTCTTA GTTAGTGTGT TTGTGGACTT TGTGGTGTAT <<<<<<<< ONW1-R 241 TGAGTGAATT TGTGTGTGTG TGTGTGTGTG AA

B: nt 001 GGTGGGACCT CCATGCGCA* CAGCGTCACT CA*CAGCGTC ACTAACACAG CGTCAAACC OnCAOl

001 * --A--A A*- Allele ZA2-1

001 * --A--G AC- Allele ZA2-2 001 T ********** ********** * AC- Allele ZA^-1

001 T ********** ********** * CC- Allele ZA1-2 001 * ********** ********** * A*- Allele ZA^-3 >>>>>>>>>> >>>>>>>> 0NZ-M1F CA RCGCAY CARCGTCACT CAYCARCGTC ACTAACA CARCGTCA = consensus repeats

061 TTTTTCCCAT GCCACTCACC ATGTTCCAAC AAGTGTCGGT ACCTCGAAGA CGCCCCCAT OnCAOl

061 A- - ZA2-1

061 A- - ZA2-2

061 A- - ZAX-1 061 A- - ZAI-2 061 T- - ZAi-3 <<<<<<<<< <<<<<<<<< ONZ-M1R Table 5.1. North American O. nubilalis maie ONZl genotypic, and heterogametic female ONWl and ONZl haplotype frequencies. WA3, WA4j and WA5 represent ONWl allele with three, four, and five GAAAAT repeats, respectively. ZAI = ONZl allele with one CAYCARCGTCACTAA consensus repeat, and ZA3 = ONZl allele with three CAYCARCGTCACTAA consensus repeats.

O. nubilalis genotypes from Z - and W-linked microsatellite markers (ONZl & ONWl) Ostrinia nubilalis S ? ID Subpopulations Pt N ZiAIZAI Za,Za2 ZA2ZA2 N Za,Wa3 ZAJWA4 ZA,WA;, Za3Wa3 Za3WA4 ZA3WA5 1 Brookings, SD6 UZ 36 0.861 0.139 0.000 23 0.217 0.749 0.000 0.044 0.000 0.000 2 South Shore, SD6 uz 31 0.968 0.000 0.032 65 0.215 0.769 0.000 0.015 0.000 0.000 3 Lamberton, MN3 UZ 24 0.875 0.402 0.083 20 0.050 0.950 0.000 0.000 0.000 0.000 4 Lamberton, MN3 BZ 29 0.862 0.035 0.103 26 0.231 0.769 0.000 0.000 0.000 0.000 5 Kanawa, IA BZ 48 0.979 0.021 0.000 45 0.089 0.733 0.178 0.000 0.000 0.000 6 Hubbard, IA BZ 46 0.913 0.022 0.065 38 0.184 0.526 0.290 0.000 0.000 0.000 7 Crawfordsville, IA BZ 40 0.947 0.026 0.026 47 0.128 0.872 0.000 0.000 0.000 0.000 8 Mead, NE5 BZ 48 0.917 0.063 0.021 40 0.225 0.750 0.025 0.000 0.000 0.000 9 Garden City, KS4 MZ 47 0.915 0.085 0.000 61 0.000 0.967 0.016 0.016 0.000 0.000 10 Columbia City, IN BZ 48 1.000 0.000 0.000 46 0.087 0.714 0.196 0.000 0.000 0.000 11 Franklin Co., IN2 BZ 48 1.000 0.000 0.000 37 0.027 0.757 0.216 0.000 0.000 0.000 12 Newark, DE1 BE 48 0.854 0.083 0.063 0 NA NA NA NA NA NA 493 458 21 14 448 59 351 38 3 0 0

O. furnicalis NA 38 0.947 0.000 0.053 41 0.000 0.871 0.129 0.000 0.000 0.000 O. pentitalis NA 1 0.000 0.000 0.000 0 0.000 1.000 0.000 0.000 0.000 0.000 104

Table 5.2. Male AMOVA and FsT, and female AMOVA and é^T values. A) Test of O. nubilalis geographic variation assuming four South Dakota and Minnesota (north latitude) subpopulations are subdivided from the remainder of the North American O. nubilalis population. B) Test of voltinism variation (univoltine moths from South Dakota and

Lamberton, Minnesota, compared to bivoltines from the remainder of the North American population).

A) Regional variation

r? ONZl Genotypes $ ONWl & ONZl Haplotypes Sum of Variance %of Sum of Variance % of df Squares Component variation df Squares Component variation Among groups 1 0.55 0.0014 Va 3.78 1 4.14 0.0090 Va 4.02 Among subpops 9 0.50 0.0002 Vb 0.63 9 19.97 0.0516 Vb 23.09 within groups Within subpops 879 31.65 0.0360 Vc 95.59 437 71.23 0.1630 Vc 72.89 Total 889 32.70 0.0376 100.00 447 36.34 0.2236 100.00

Fixation Indices 95% CI Fixation Indices 95% CI Fsc (Fis) 0.007 0.037+0.005 @sc(&is) 0.241 0.000±0.000 Fst 0.044 0.022+0.003 0.271 0.000+0.000 Fct (FlT) 0.038 0.004±0.002 0{ ( 0\ i ) 0.040 0.106+0.014

B) Voltinism variation

r? ONZl Genotypes $ ONWl & ONZl Haplotypes Sum of Variance %of Sum of Variance % of df Squares Component variation df Squares Component variation Among groups 1 0.18 0.0003 Va 0.83 1 0.82 -0.0136 Va -6.44 Among subpops 9 0.87 0.0008 Vb 2.01 9 23.29 0.0614 Vb 29.11 within groups Within subpops 879 31.65 0.0360 Vc 97.16 437 71.23 0.1630 Vc 77.33 Total 889 32.70 0.0376 100.00 447 36.34 0.2236 100.00

Fixation Indices 95% CI Fixation Indices 95% CI Fsc (F,s) 0.020 0.000+0.015 M4s) 0-273 0.000+0.000 Fst 0.028 0.000+0.014 6b 0.227 0.000+0.000 Fct (Fit) 0.008 0.010±0.002 (\'x ( 6>it) -0.064 0.106+0.014 105

CHAPTER 6. SEQUENCE VARIATION IN THE CADHERIN GENE OF OSTRINIA NUBILALIS'. A TOOL FOR FIELD MONITORING

Brad S. Coates, Douglas V. Sumerford, Richard L. Hellmich, and Leslie C. Lewis

Paper published: Insect Biochemistry and Molecular Biology 35:129-139, 2005

Abstract

Toxin-binding proteins of insect midgut epithelial cells are associated with insect resistance to Bacillus thuringiensis (Bt) Cry toxins. A 5378 nt cDNA encoding a 1717 amino acid putative midgut cadherin-like glycoprotein and candidate CrylAb toxin-binding protein was characterized from Ostrinia nubilalis. Intraspecific alignment of partial O. nubilalis cadherin gene sequences identified variance within proposed CrylA toxin binding region 2 (TBR2),

1328IPLQTSILVVT[I/V] N1340, and flanking CrylA toxin binding region 1 (TBR1),

861DIEIEIIDTNN871. DNA sequence and PCR-RFLP detected single nucleotide polymorphism (SNP) between cadherin alleles, and pedigree analysis demonstrated

Mendelian inheritance. A population sample from Mead, Nebraska showed allelic polymorphism. These assays may be useful for linkage mapping and field surveillance of wild populations of O. nubilalis.

Keywords: Cadherin, Bacillus thuringiensis receptor, midgut cDNA

Introduction

Resistance to insecticides commonly evolves in agricultural fields when insecticides are frequently applied at high doses to control insect pests (Georghiou and Lagunes-Tejeda

1991). The western corn rootworm (Diabrotica sp.; Miota et al. 1998; Zhu et al. 2001) evolved field resistance to carbaryl insecticides. Indian mealmoth (Plodia interpunctella; 106

McGaughey, 1985) and diamondback moth (Plutella xylostela\ Tabashnik et al. 1990)

evolved resistance to insecticides with toxins of the bacterium Bacillus thuringiensis var.

kurstaki. Most of the documented instances of insect resistance involve a small number of

genes (Roush and McKenzie 1987). Insecticide resistance occurred by a single amino acid

change in acetylcholinesterase, the target of organophosphate and carbamate insecticides in house fly (Williamson et al. 1996), and the sodium channel peptide targeted by pyrethroids in cockroach (Miyazaki et al. 1996). Similarly, cyclodiene resistance occurs by a single nonsynonymous point mutation in the Drosophila y-aminobutyric acid (GABA) receptor

(ffrench-Constant et al. 1993).

European corn borer, Ostrinia nubilalis (Lepidoptera Crambidae), a lepidopteran pest of maize, has been controlled with foliar applications of the bacterium B. thuringiensis (Bt) var. kurstaki. Incorporation of the B. thuringiensis CrylAb toxin gene into maize germplasm

(Koziel et al. 1993) results in expression that controls O. nubilalis larvae (Walker et al.

2000). High selection pressure and adaptation to transgenic toxins by O. nubilalis larvae raises concerns regarding sustainability of Bt technology. Commercial hybrid maize expressing CrylAb toxins were grown on 29% of 2003 United States corn acres (22.9 million acres; USDA-NASS, 2003). Resistance has not been detected in the field; but moderate resistance was found in O. nubilalis laboratory colonies fed artificial Bt containing diets (Bolin et al. 1999; Huang et al. 1999; Chaufaux et al. 2001).

Modification of midgut receptor proteins that bind Cry toxins may reduce susceptibility of insects. Ligand blots demonstrate that CrylAb binds to a 220-kDa cadherin- like glycoprotein, and 145-kDa and 154-kDa aminopeptidase (APN) isoforms in O. nubilalis

(Hua et al. 2001). A Manduca sexta 210-kDa E-cadherin-like peptide bound activated 107

(trypsinized) CrylA toxin (Francis and Bulla, 1997). Midgut expressed cadherin-like cDNAs were isolated fromM. sexta (Vadlamudi et al. 1995), Bombyx mori (Nagamatsu et al.

1998a), Heliothis virescens (Gahan et al. 2001), and Pectinophora gossypiella (Morin et al.

2003). Furthermore, a cadherin knockout from a transposon insertion resulted in 40,000-fold resistance in the H. virescens YHD2 colony (Gahan et al. 2001). Segregation of alleles at a cadherin locus was associated with resistance in P. gossypiella strain AZP-R (Morin et al.

2003). CRYl A domain II binds lepidopteran midgut epithelial receptors (deMaagd et al.

2001) by interacting with two cadherin motifs (Gomez et al. 2003). Cadherin CrylA toxin binding region (TBR1), 865NITIHITDTNN875, was identified on M sexta gene Bt-Ri and implicated in binding loop 2 of CRYl A domain II (Gomez et al. 2001; 2002a; 2002b). A second binding region (TBR2) was identified in B. mori cadherin repeat 9 (CR9; Nagamatsu et al. 1999) and M. sexta cadherin CRI 1 (Dorsch et al. 2002). Cadherin TBR2 was narrowed to 12 amino acids of Bt-Ri, 1331IPLPASILTVTV1342, and predicted to interact with loop a-8 of CRYl A domain II (Gomez et al. 2003). Effects of cadherin toxin binding region variation on resistance traits are unknown.

The extracellular domain of cadherin peptide also has been implicated as a CrylAb binding receptor in the O. nubilalis midgut (Hua et al. 2001). Candidate CrylAb binding receptor genes have not been reported in O. nubilalis. In this paper we report research that isolated a full-length midgut cadherin-like cDNA from 5th instar O. nubilalis larvae, determined allelic variation (single nucleotide polymorphism; SNPs) in and near two putative

CrylAb toxin-binding regions (TBR1 and TBR2), and characterized Mendelian segregation of SNPs among pedigrees by PCR-RFLP. Additionally, in a preliminary field screening 108 experiment we estimated cadherin allele variation in a susceptible population of O. nubilalis near Mead, Nebraska.

Materials and Methods

Cadherin Alignment and Phylogeny

A 3048 residue consensus alignment was constructed among 11 lepidopteran cadherins, excluding O. nubilalis, using Align X software (Informax, San Francisco, CA; gap

penalty = 5). Regions of peptide similarity were identified and corresponding cDNA sequence used to design degenerate 3' RACE oligonucleotides with Primer3 (Rozen and

Skaletsky 1998; see primer Cad3pR in section 2.2). Twelve lepidopteran cadherin amino acid sequences, including O. nubilalis, were placed into a single phylogeny using programs

from the PHYLIP package (Felsenstein 1989). One thousand bootstrap resampling steps were produced by the SeqBoot program, parsimony trees were generated using AAPars, a strict consensus tree was estimated from all possible phylogenies with CONSENSE, and was viewed using TreeView (Page 1996).

Complementary DNA (cDNA) Synthesis and Cloning

Ten dissected larval O. nubilalis midguts were bulked, ground to powder in liquid nitrogen, and RNA extracted with RNeasy extraction kits (Qiagen, Valencia, CA) according to manufacturer instructions. First strand cDNA synthesis used 0.5 fiM of poly(T) adapter

(PT-AD; GOT GTA ATA CGA CGG CCT GGA ATT CTT TTT TTT TTT TTT TTT T), 2

|ig of total RNA, 40 U AMV reverse transcriptase (Promega, Madison, WI) in a 20 |_il reaction volume incubated at 42 °C for 1 h. Primer Cad3pR (TMA ACA ACA CCC CTC 109

CYA CKC T) was designed using Printer] (Rozen and Skaletsky, 1998) to anneal transcript sequence corresponding to a NNTPPLT amino acid sequence, which was conserved among lepidopteran cadherins. Hot start PCR of first strand cDNA used 5 pmol each of degenerate primer Cad3pR and poly(T) adapter primer 1 PT-Adcl (GTG TAA TAC GAC GGC CTG

G), 1.0 U Tli proofreading polymerase (Promega), 100 pM dNTPs, 1.5 mM MgCla, and 2.5 pi of 10X thermal polymerase buffer (Promega) in a 25 jo.1 reaction volume. A PTC-100 thermocycler (MJ Research, Watertown, MA) cycled 40 times at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 4 m. Amplification was confirmed and fragment size estimated by electrophoresis of 10 pi PCR product on a 10 cm 1.5 % agarose IX tris-borate EDTA gel containing 0.5 pg/ml ethidium bromide. PCR product was ligated using the pGEM-T easy cloning system (Promega) by overnight incubation at 4 °C, and was used to transform 80 pi

E. coli SURE (Stratagene). DNA sequencing of plasmid inserts used dye-terminator cycle sequencing (DTCS) quick-start kits (Beckman-Coulter, Fullerton, CA) and fragments separated on a CEQ8000 capillary electrophoresis system (Beckman-Coulter). Identity of insert sequence was checked by BLAST search, and transcript-specific 5' RACE primers designed (Primer3; Rozen and Skaletsky, 1998).

Oligonucleotide primer Cad5pR (CTG CAT CGA TCG CCT CGT T) was designed from 3' RACE cDNA sequence. First Choice RLM RACE kit (Ambion) was used to isolate

5' cDNA end of the O. nubilalis cadherin transcript according to manufacturer instructions;

RLM-RACE template was generated in 20 pi reactions with 1 pg of midgut total RNA.

Subsequent hot start PCR used 1.0 U Tli proofreading polymerase (Promega), 200 pM dNTPs, 1.5 mM MgCl%, and 2.5 pi of 10 x thermal polymerase buffer (Promega), 1 pi RLM 110 mRNA template, 5 pmol each of degenerate primer Cad5pR, and 1 pi RLM-RACE 5' adapter outer primer (Ambion; GCT GAT GGC GATG AAT GAA CAC) in a 25 pi reaction. A

PTC-100 thermocycler (MJ Research) cycled 40 times at 95 °C for 30 s, 58 °C for 30 s, and

72 °C for 4 m. RT-PCR products were ligated, cloned, and sequenced as described previously.

Polymorphism with Putative Cadherin CrylAb Toxin Binding Domains

Two putative CrylA toxin-binding domains, 861DIEIEIIDTNN871 (TBR1) and

1328IPLQTSILVVTV1339 (TBR2), were identified from a single 1717 residue O. nubilalis midgut cDNA open reading frame (ORE). TBR1 and TBR2 were PCR amplified using primers OnCadTBRl-F (CAA ATT GAG GTG GAT GCG AA) with OnCadTBRl-R (TGT

CAC GGT CGT GGG AGT ACA), and OnCadTBR2-F (TYT CTT CAA CAG ACG GCA

ACA A) with OnCadTBR2-R (ACC CTG ACG GTA AGC AAT TCC C). PCR products were referred to as O. nubilalis CrylA toxin binding region 1 (OnTBRl) and 2 (OnTBR2), and amplified using 2.5 mM MgCl], 50 pM dNTPs, 7.5 pmol of each primer, 0.9 U Taq

DNA polymerase (Promega), and 100 ng of DNA template (equal mix of 10 individual DNA samples) in a 12.5 pi reaction. PTC-100 thermocycler conditions used 95 °C for 3 m, followed by 40 cycles of 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 20 s. OnTBRl and

OnTBR2 were ligated into pGEM-T vector (Promega), and used to transform E. coli SURE

(Stratagene). Plasmid inserts were sequenced as described previously. Sequence data were imported into Vector NTI Suite 7.0 (Informax, San Francisco, CA) and multiple sequence alignments were made using AlignX software (Informax; gap penalty = 5). Ill

A third PCR product (OnTBR2-s) used primer OnCadTBR2-F with OnCadTBR2-R2

(CTY AGC TTT TGC ASG AAG AAA G) to amplify an 0. nubilalis cadherin gene region containing 1328IPLQTSILVVTV1339. The OnTBR2-s PCR product is smaller and eliminates length polymorphism contained within an intron spanned by the original OnTBR2 product, thus facilitated ease of SNP genotyping. Polymorphic restriction endonuclease cleavage sites from OnTBRl, Ndell, Rsal, Taql, and Tsp509l, and OnTBR2, Cfol, Haelll, and HpyCRAlll, were identified from DNA sequence alignments. All endonucleases were purchased from

Promega, except 7!sp509I and HpyCRAlll (New England BioLabs). PCR-RFLP reactions of

fragments OnTBRl and OnTBR2-2 used PCR conditions described previously scaled to 25

pi. Cfol, Ndell, Rsal, Taql, and Tsp509l digests included 6.0 pi of PCR product, 2.5 pi lOx

Buffer, 0.1 mg/pl BSA, and 0.25 U of enzyme in 25 pi. OnTBR2-2 Haelll and HpyCHAHl double digestions used 0.5 U of each enzyme. Reactions were incubated at 37 °C or 60 °C

(7sp509I) for 8 to 14 h. Entire digest volumes were loaded onto 1.0 mm by 16 cm 6% polyacrylamide (19:1 acrylamide: bisacrylamide) 0.5X TBE gels, and separated at 140 V for

6 h. A 25 bp step-ladder (Promega) was used for size comparison. Gels were stained with ethidium bromide, and digital images taken under UV illumination on a PC-FOTO/Eclipse

Documentation System (Fotodyne, Hartland, WI). Allele frequencies for cadherin loci were estimated from 96 O. nubilalis adults.

Cadherin Allele Frequencies and Mendelian Inheritance

Over 300 adult O. nubilalis were collected in a single light trap at a non-Bt corn field near Mead, Nebraska in August 2002 (contributed by John Seymour, University of

Nebraska), and were samples stored separately in 1.5 ml microcentrifuge tubes at - 20 °C. A 112

subsample of 92 individuals was screened with previously described PCR-RFLP protocols,

and used to provide baseline estimates cadherin allele frequencies in a given population.

Two paired matings of O. nubilalis from the USDA-ARS, Corn Insects and Crop

Genetics Research Unit laboratory colony were used to establish Fi families. The Fj progeny

were sib-mated to produce F2 larvae. F2 larvae were reared to adults and used to estimate

genotype frequencies for cadherin loci. Larvae were reared on artificial diet (Guthrie 1987)

and adult samples were stored separately in 1.5 ml microcentrifuge tubes at - 20 °C prior to

DNA isolation. DNA from field and pedigree samples was extracted from adult thorax tissue

using methods described by Coates and Hellmich (2002). Genotyping by PCR-RFLP was

performed as described previously. The goodness-of-fit of observed genotypic distributions

during the F2 generation were compared to expectations based on Mendelian inheritance.

Results and Discussion

Ostrinia nubilalis Cadherin Identification

A 220-kDa cadherin-like peptide from O. nubilalis was identified as a midgut

CrylAb receptor (Hua et al. 2001). We isolated a 5378 nt O. nubilalis midgut-expressed

mRNA (GenBank accession no. AY612336) similar to lepidopteran cadherin transcripts (H.

armigera AF519180, BLAST score = 92, E-value 10~14; B. mori AB041509, BLAST score =

54, E-value 0.003). A single 5154 nt open reading frame (ORF) encoded a putative 1717

amino acid polypeptide (Fig. 6.1). Protein Database (PDB) search with PredictProtein (Rost,

1996) demonstrated high similarity (S) and identity (IDE) of the putative O. nubilalis peptide to B. mori BtR175 (S = 73%, IDE = 61%; Nagamatsu et al. 1998b), L. dispar BTR-CAD (S =

72%, IDE - 60%; GenBank accession no. AF317621), M. sexta Bt-Rl (S = 58%, IDE = 113

72%; Vadlamudi et al. 1995) and H. virescens BtR-4 (S = 58%, IDE = 72%; Gahan et al.

2001). A parsimony-based phylogeny used a consensus 3048 amino acid alignment of 12 lepidopteran cadherins including O. nubilalis and indicated three main divisions: cadherins from superfamilies Noctuoidae, Bombycoidae and Sphingoidea, and Pyraloidea and

Gelchioidea (Fig. 6.2). Internal branch support at internal nodes is greater than 85% based on 1000 bootstraps, but terminal nodes among B. mori alleles may show homoplasy due to homologous recombination. Placement of the O. nubilalis ORF within the cadherin peptide phylogeny and high peptide similarity to other lepidopteran cadherin, suggests the O. nubilalis midgut cDNA (GenBank accession no. AY612336) encodes a cadherin-like peptide.

Cadherin-like motifs were identified within the derived O. nubilalis polypeptide. A

ProSite database search identified a 1402 amino acid long extracellular domain, a 21 residue signal peptide (SIG), transmembrane region (TMR), and cytoplasmic domain (CPD; Fig.

6.1). Eleven consensus cadherin repeat motifs ([LIV]-x-[LFV]-x-D-x-N-D-[NH]-x-P) were identified by PredictProtein (Rost 1996). The 1717 amino acid O. nubilalis cadherin-like peptide has predicted molecular weight (192 kDa) similar to B. mori and M. sexta homologs

(Compute pI/Mw program; Bjellqvist 1994). Cadherins are glycoproteins involved in calcium-dependent cell-cell adhesion (Takeichi 1990). There are 13 predicted O. nubilalis cadherin peptide N-glycosylation sites (NP[S or T]P; Gavel and von Heijne 1990; Fig. 6.1).

Difference between polyacrylamide mobility (220-kDa; Hua et al. 2001) and estimated 192 kDa may be due to posttranslational modifications. Predicted isoelectric point (pi) of the O. nubilalis cadherin peptide (4.23; Compute pFMw program; Bjellqvist 1994) indicates 114 solubility in alkaline midgut environments, which is congruent with required extracellular

domain function.

Polymorphism with Putative Cadherin CrylAb Toxin Binding Domains

Two putative O. nubilalis cadherin peptide CrylA toxin-binding regions

861DIEIEIIDTNN871 and 1328IPLQTSILVVTV1339 were identified (Fig. 6.1) that were

identical to 7 of 11 homologous M. sexta Bt-Ri TBR1 residues (865NITIHITDTNN875;

Gomez et al. 2001) and 9 of 12 homologous M. sexta Bt-Ri TBR2 residues,

1331IPLPASILTVTV1342 (Gomez et al. 2003). A cadherin alignment of amino acid sequence

(Fig. 6.3) indicates five TBR1 (I868, 1870, D872, and N875) and two TBR2 amino acids are fixed between all species (L1339 and T1341; with respect to M. sexta Bt-Ri). Interspecies

comparisons reveal little conservation of the cadherin toxin-binding region. Variability in

cadherin sequences may allow flexibility of CrylAb toxin binding to midgut receptors or a

generalized protein-protein interaction in CrylA toxin mode of action.

Intraspecific DNA sequence alignment detected variation among cloned OnTBRl

(Fig. 6.4) and OnTBR2 (Fig. 6.5) cadherin gene PCR products, containing 214 to 232 and

222 to 232 nt introns, respectively. No insertions or deletions were observed in coding

sequence (exons). In addition to intron length variation, 31 OnTBRl SNPs were observed (n

= 0.02707 (Nei 1987); 0 = 0.02809 (Tajima 1996)); 4 of 8 of these were synonymous

changes in exons (Fig. 6.4). Thirty-one OnTBR2 SNPs were identified (n = 0.02410; 0 =

0.02490); 13 of 17 of these were synonymous changes in exons (Fig. 6.5). Eight SNPs were

identified within restriction endonuclease recognition sites. Four polymorphic restriction sites (Ndell, Rsal, Taql, and Tsp509A\) were identified from OnTBRl (Fig. 6.4); and four 115 sites (Cfol, Haelll, //pjCHVIII and Mspï) were identified from OnTBR2 (Fig. 6.5).

Subsequent PCR-RFLP assays used PCR product OnTBR2-s for 1328IPLQTSILVVTV1339 region SNP detection. One SNP within a ///ryCHVIII recognition site represents a nonsynonymous peptide change (1328IPLQTSILVVT[I/V] N1340). Alternate amino acids (I or

V) both have short chain aliphatic side groups and may not affect regional charge or peptide structure. Remaining PCR-RFLP indicate nonsynonymous changes of cadherin alleles outside 861DIEIEIIDTNN871 and I328IPLQTSILVVT[I/V] N1340 (Figs. 4, 5).

The nucleotide substitutions in the cadherin gene and PCR-RFLP assays provide a means for genotyping O. nubilalis, mapping of resistance traits, and characterizing populations. Three cadherin alleles (rl, r2, and r3) in Cry1 Ac resistant P. gossypiella strain

AZP-R differed by block deletion or truncation (Morin et al. 2003), and shared no fixed peptide sequence differences compared to alleles from a susceptible line. Resistant allele r2 had a truncation deletion that eliminated homologous P. gossypiella TBR1

(862DNVINIVDINNK873) and TBR2 (1341PLPGSLLTVTVT1352); and allele rl had a nonsynonymous change directly flanking TBR1 (R861) compared to of susceptible and r3 alleles (G861). Morin et al. (2003) also established a correlation between cadherin resistance alleles and increased P. gossypiella larval weight among individuals from a Texas field sample (TX-01) reared on diet containing 1 pg Cry1 Ac/ml. Recessive Bt resistant alleles were independently selected for in P. gossypiella strain APHIS-98R (Tabshnik et al. 2004), suggesting utility of fielding surveillance to monitor the development of resistance. 116

Mendelian Inheritance and Cadherin Allele Frequencies

Mendelian inheritance of 0. nubilalis cadherin alleles was established within F2 pedigrees, Ped31 and Ped62. Genotype ratio expected among F2 progeny was determined assuming Mendelian segregation of parental alleles. The observed genotype did not significantly deviate from expected ratios in either pedigree for assays in which parentals

2 were polymorphic (% test; Table 6.1). Adherence of F2 progeny to expected Mendelian ratios suggests null alleles are not present. PCR-RFLP assays are therefore appropriate as markers for linkage mapping or population genetic studies (Pemberton et al. 1995).

Molecular screening may detect geographic regions of increased insecticide resistance levels and are increasingly being incorporated into resistance diagnostics (Brogdon and McAllister 1998). Molecular assays have been developed to detect resistance genes in insect populations (Steichen et al. 1994; Field et al. 1996), and screening known resistance alleles in P. gossypiella may show promise for population surveillance (Morin et al. 2003;

Tabashnik et al. 2004). In a preliminary surveillance experiment we used PCR-RFLP to estimate frequencies of O. nubilalis cadherin alleles (SNP haplotypes) in a population of susceptible adults collected near Mead, Nebraska. Three to six alleles were observed per

PCR-RFLP assay (OnTBRl Rsal, Taql, and 7sp509I, and OnTBR2-s Cfol and Haelll with

HpyCYlA III) and 21 unique allelic combinations (genotypes) were observed. Frequency of cadherin alleles were defined from individual restriction assays and ranged from 0.012 to

0.455 (Table 6.2).

One or more genes may mediate lepidopteran resistance to CrylA toxins.

Aminopeptidase was implicated as a major midgut receptor (Knight et al. 1994; Gill et al.

1995), followed by cadherin (Vadlamudi et al. 1995). Screening P. xylostella for survival on 117

CrylA and Cry IF toxins suggested a single gene imparts cross-resistance (Tabashnik et al.

1997). This was corroborated when a major autosomal locus inherited as a recessive trait was identified (Meckel et al. 1999). Cadherin was implicated as a resistance factor by phenotypic association in P. gossypiella (Morin et al. 2003) and transposon knockout in H. virescens (Gahan et al. 2001). For Spodoptera litura, transcript knockdown by RNA interference (RNAi) showed the role of aminopeptidase in Bt resistance (Rajagopal et al.

2002). Inheritance of resistance in an O. nubilalis strain selected for survival on Dipel® Bt formulations was autosomal and incompletely dominant (Huang et al. 1999). Additive gene action was predicted to mediate Bt resistance in H. zea (Burd et al. 2003); and two copies of cadherin resistance alleles (rl, r2, or r3) were required for resistance in P. gossypiella (Morin et al. 2003).

To manage resistance effectively, it is necessary to understand the genetic mechanisms of resistance (Gould and Tabashnik 1998; Caprio et al. 2000) and monitor insect pests of transgenic-insecticidal cultivars for changes in their tolerances of Bt toxins (Gould et al.

1997; Andow and Alstad 1998). To do this, it is necessary to track the evolutionary changes of resistance alleles in pest populations. The full-length mRNA, partial-genome sequence, and molecular markers developed in this study have downstream applications that will aid in the determination of cadherin involvement in potential O. nubilalis resistance to CrylAb toxin. These markers are appropriate for incorporation into linkage mapping studies.

Laboratory colonies of Ostrinia nubilalis that exhibit moderate resistance to CrylAb toxin in artificial diet (Bolin et al. 1999; Huang et al. 1999; Chaufaux et al. 2001), and diagnostic bioassays can discriminate phenotypes (Marcon et al. 2000). OnTBRl and OnTBR2 PCR-

RFLP markers show Mendelian inheritance (Table 6.1), which is required for genetic 118

mapping procedures (Pemberton et al. 1995). F2 pedigrees in conjunction with the bioassays can be used to determine if the segregation of the moderate O. nubilalis resistance phenotype is associated with cadherin allele segregation using OnTBRl or OnTBR2 PCR-RFLP assays.

A similar protocol was used to associate cadherin alleles rl, r2, and r3 with Cry1 Ac resistance in the P. gossypiella AZP-R strain and field collections (Morin et al. 2003).

Association of cadherin alleles with distinct phenotypic traits is a prerequisite for effective field surveillance, but marker polymorphism determines how effectively alleles are differentiated. Our cadherin loci will also be incorporated into linkage maps of other marker types (AFLP and microsatellites) to better examine cause and effect between resistant phenotypes and cadherin loci. This will help pinpoint the location of putative resistant loci and ask if their location is in the vicinity of our cadherin maker loci.

Polymorphisms among O. nubilalis cadherin alleles at Mead, Nebraska for OnTBRl and

OnTBRl indicates that native genetic variation is present. The native genetic variation for cadherin loci can be incorporated into resistance monitoring or surveillance programs, pending allelic association with potential field resistance traits via bioassays. Studies are

currently underway with O. nubilalis to map the genomic position of the cadherin locus. We

will use the information obtained not only to better understand inheritance of CrylAb toxin

resistance, but also examine the appropriateness of allelic association with phenotypic traits

for potential incorporation into field monitoring or surveillance programs.

Acknowledgements

This research was a joint contribution from the USDA, Agricultural Research Service and the

Iowa Agriculture and Home Economics Experiment Station, Ames, IA (project 3543). This 119 article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation by USDA or Iowa State University for its

use.

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Figure 6.1. Ostrinia nubilalis 1717 residue long cadherin-like peptide sequence

(GenBank accession no. AY612336) with a predicted 21 amino acid signal peptide (SIG), 11 cadherin repeats (CRI to CR11), 13 extracellular glycosylation sites, membrane proximal region (MPR), transmembrane region (TMR), and cytoplasmic domain (CPD). Putative glycosylation sites are underscored by triangles in diagram and sequence. Two potential O. nubilalis Bacillus thuringiensis CrylA toxin binding regions with homology to those proposed by Gomez et al. (2002; 2003) are underscored.

MPR SIG TMR CR1 C%l CR5 |6rëg| CRloK^ïjOfl CPD TBR1 | TBR2 1

SIG -> 0001 BBSS!IEBSHH! INQRCSYIIA IPRPETPELP PIDYEGKSWS EQPLIPGPTR CR1 -> 0061 EEVCMENFLP DQMIQV1YME EEIEGDV1IA KLNYQGSNTP VLSIMSGQPR AQLGPEFRQN

0121 ÈADGQWSIIVI TQRQDYETAT MQSYVFSIQV EGESQAVLVA LEIVNIDDNP PILQWSACV CR2 -» 0181 IPEHGEARLT DCVYQVSDRD GEISTRFMTF RVDSSRAADE SIFYMVGEYD PSDWFNMKMT

0241 VGINSPLNFE TTQLHIFSVT ASDSLPNNHT VTMMVQVENV ESRPPRWVEI FSVQQFDEKT CR3 -> 03 01 NQSFSLRAID GDTGINRAIN YTLIRDDADD FFSIJE»IEPG AILHVTEIDR DKLERELFNL A A CR4 -> A 0361 TIVAYKSTDA IMEETPMTLN FNEEEGFHDR A 0421 DLGENAQYTV ELEDVFPPGA ASAFYIAPGS GYQRQTFIMG TINHTMLDYE DVIFQNIIIK CR5 "> A 04 81 VKAVDMNNAS HVGEALVYVN LINWNDELPI FEESSYSASF KETVGAGFPV ATVLALDRDI A 0541 DDWVHSLMG NAVDYLFIDE STGEIFVSMD DAFDYHRQNT LFVQVRADDT LGDGPHNTVT CR6 "> 0601 TQLVIELEDV NNTPPTLRLP RSTPSVEENV PEGYEISREI TATDPDTSAY LWFEIDWDST

0 661 WATKQGRETI PTEYVGCIVI VTIYPTEGNR GSAIGRLWQ EIRDNVTIDF EEFEMLYLTV CR7 -> A 0721 RVRDLNTVIG DDYDEATFTI TIIDMNDNAP IFANGTLTQT MRVRELAASG TLIGSVLATD A OnTBRl 07 81 IDGPLYNQVR YTIQPRNNTP EGLVKIDFTT GQIEVDANEA IDADEPWRFY LYYTVIASDE TBR1 CR8 -> 0841 CSLENHTEGP PDPNYFEVPG JSEESBH ÎIKVPEPLTEK FNTTVYVWEN ATSGDEVVQL A A A 126

0901 YSHDRDRDEL YHTVRYTMNF AVNPRLRDFF EVDLDTGRLE VHYFGDEKLD RDGDEPTHTI

CR: ' 0961 FVNFIDNFFS DGDGRRNQDE VEIFWLLDV NDNAPEMPLP DELRFDVSEG AVAGVRVLPE

1021 IYAPDRDEPD TDNSRVGYGI LDLTITDRDI EVPDÊFTMIS IENKTGELET AMDLRGYWGT A CR10 -> 1081 YEIFIEAFDH GYPQQRSNET YTLVIRPYNF HHPVFVFPQP DSVIRLSRER ATEGGVLATA A 1141 ANEFLEPIYA TDEDGLHAGS VTFHVQGNEE AVQYFDLTEV GAGENSGQLI LRQLFPEQIR

1201 QFRITIRATDG GTEPGPLWT DVTFSWFVP TQGDPVFSEN AATVAFFEGE EGLHESFELP CR11 OnTBR2 1261 QAEDLKNHLC EDDCQDIYYR FIDGNNEGLFV LDQSRNVIS LAQELDREVA TSYTLHIAAS TBR2 MPR 1321 NSPDATGifli MWSMMIMMMflN VREANPRPIF EQDLYTAVIS TLDSIGRELL TVRASHTEDD

1381 IITYTIDRAS MQLDSSLEAV RDSAFALHAT TGVFSLNMQP TASMHGMFEF DVIATDTASA

1441 IDTARVKVYL ISSONRVTF$ FDNQLETVEQ NRNFIAATFS TGFNMTGNID QWPFSDSSG A 1601 VAQrâTtEVR AHFIimmPV QAQEVEAVRS DTVLLRTIÙL MLSTNSLVLQ DLVTGDTPTL TMR "> CPD "> 1661 GEESMOIAVY ALAALSAVLG FLCLVLLLAL FCRTRALNRQ LQALSMTKYG SVDSGLNRAG

1721 LAPGTNKHAV EGSNPMWNEA IRAPDFDAIS DASGDSVLIG lEDMPQFRDD YFPPGDTDSS

1781 SGIVLHMGEAT DNKPVTTHG NNFGFKSTPY LPQPHPK 127

Figure 6.2. Lepidopteran cadherin phytogeny. Parsimony-based phytogeny of 12 lepidopteran cadherin peptides generated from a 2048 amino acid consensus alignment.

Level of bootstrap support is indicated at each node (N=1000). Branch termini labeled with

GenBank accession nos. and respective species indicated as follows, Bm {Bombyx mori), Ms

{Manduca sexta), Cs {Chilo suppressalis), On {Ostrinia nubilalis), Pg {Pectinophora gossypiella), Ld {Lymantria dispar), Hv {Heliotis virescens), and Ha {Helicoverpa armigera). Drosophila cadherin Cad99C served as outgroup to root the tree.

AB026260Bm 611 "D« AB041508Bm 0 1000 1 AB041509Bm oE cû 593

1000 ' AB041510Bm

tdo> ' AY094541 Ms •o ÔUi 1000I c ic AF319873MB a 851 CZ) CO AY118272CS "Oa> 978 o icv 987 -AY6123360n A> 974 o (0 - AY198374Pg a> "O 387 0 - AF317621Ld 1

1000 AF367362HV

1000

AF519180Ha

Dmeicadsec Fig. 6.3. Variation in lepidopteran cadherin toxin binding regions (TBR1 and 2). Conservation in proximity to putative cadherin CrylA toxin binding regions 861DIEIEIIDTNN871 (panel A) and 1328IPLQTSILVVT[I/V] N1340 (panel B) across lepidopteran lineages is shown. Species abbreviation and GenBank accession nos. for aligned peptides are as in Fig. 6.2. Amino acid residues conserved with O. nubilalis (On) cadherin (GenBank accession no. AY612336) are highlighted. Alternate O. nubilalis amino acid residues (Onalt) are shown in a consensus of all sequenced alleles (Figs. 3 and 4).

A) Homologous lepidopteran amino acid sequence spanned by PCR product OnTBRl

819 900 AY612 3 3 60n EAIDADEPWRFYLYYTVIASDECSLENHTECPPDPNYFEVPGDIEIEIIDTNNKVPEPLTEKFNTTVYVWENATSGDEWQL Onalt g g 1 N AY094541MS GAIDADTPPRFHLYYTWASDRCSTEDPADCPPDPTYWETEGNITIHITDTNNKVPQAETTKFDTWYIYENATHLDEWTL AY118272CS EAIDADVPPRYHLYYQVIASDQCLED***ECPPDPMHFNTTGYIQIEILDTNNKVPRGTLGPIQVGGAHQREITQRLPWQL AF31762lLd SAIDADTPPRYNLYYTIIASDQCYAENVEDCPPDPTYWETEGLJSIYIIDTNTJKIPQAEIELFSTTVYVWENATSGTEIETI AY198374Pg GAIDADIPPRWHLNYTVIASDKCSEENEENCPPDPVFWDTLGDNVINIVDINNKVPAADLSRFNETVYIYENAPDFTNWKI AB 026260Bm GAIDADVPRRYNLYYTWATDRCYAEDPDDCPDDPTYWETPGQVVIQIIDTNMKIPQPETDQFKAWYIYEDAVSGDEWKV AF519180Ha QAIDADEPPRQNLYYTVIASDKCDLLTVTECPPDPTYFETPGEITIHITDTNNKVPQVEDDKFEATVYIYEGADDGEHWQI AF367362HV QAIDADEPPRQHLYYTWASDKCDLLSVDVCPPDPNYFNTPGDITIHITDTNNRVPRVEEDKFEEIVYIYEGAEDGEHWQL

B) Homologous lepidopteran amino acid sequence spanned by PCR product OnTBR2-s

1287 1340 AY612 336On EGLFVLDQSRNVISLAQELDREVATSYTLHIAASNSPDATGIPLQTSILWTVN

AY094541MS EGHFGLDPVRNRLFLKKELIREQSASHTLQVAASNSPD * GGIPLPASILTVTVT AY118272CS GGYFQLDPVRNRLTLARALDQEQNRFHS IWAASNSPSATGTPLDGTTliTVTIN AF31762lLd DGHFAVNPVTNVIYLVEELDREVEETYTILVAASNSPDS*VNAliPSWTLTVTVN AY1983 74Pg YEPFDLDPVTNVIFLKSELDRETTATHWQVAASNSPTGGGIPLPGSLLTVTVT AB02 62 60Bm DGHFGLDETTNVLFLVKELDRSVSETYTLTIAASNSPT*GGIAL*TSTITITVM AF51918 0Ha GEHFRVDPRTNVLTLVKPLDRSEQETHTLIIGASDTP*NPAAVLQASTLTVTVM AF3 673 62HV MGHFAVDPQSNELFLLTPLERAEQETHTLIIGASDSP*SPAAVLQASTLTVTVM 129

Figure 6.4. Alignment of 0. nubilalis cadherin CrylA toxin binding region 1

(OnTBRl) genomic DNA sequence GenBank accession nos. AY627288 to AY627295.

Primer annealing sites are indicated by arrows showing direction. Identical nucleotides (-

) and deletions (*) are indicated with respect to uppermost reference sequence. Introns are overwritten (A) and amino acids encoded in exons are labeled using standard 21- symbol code. Polymorphic NdeII, Rsal, Taql, and Tsp5091 restriction endonuclease sites are underlined and labeled appropriately.

Primer OnCadTBRl-F !>>>>>>>>>>>>>>>>>>>> EAIDADEPWRFYLYYST V70 AYG27288 CAAATTGAGGTGGATGCGAACGAGGCGATCGATGCAGACGAACCCTGGCGCTTCTACTTGTACTACTCCG AY62 7289 A__ AY62 7290 A AY6 2 7291 A AY62 7292 A AY62 7293 T AY627294 A AY62 7295 A

71 IASDECSLENRTECPPDPSNYFE IV140 AY6 2 72 88 TCATCGCCAGCGACGAGTGCTCCCTGGAAAACCGCACGGAATGTCCTCCAGATCCCAACTACTTCGAAAT AY6272 89 C A -A- AY62 729 0 C G -A- AY62 7291 T A X- - G- AY62 7292 C G T- - G- AY62 7293 T A T-- G- AY62 7294 T G T-- G- AY62 7295 T A T~ - G- Nde II Tsp5 091

141 P G D Intron AY627288 TCCAGGCGATGTGAGTAATGAATATTACTGTCTGAAGCCTTTAAGGTTGTACGCTGTGTCATCATCTACT AY62 72 89 G A A GT C T AY62 7290 G A A GT C T 62 72 91 — — — — — — — — — — — — — — — — Q — — — — — — — x — — — — — — — — — — XT — — — — G — — — — — — — — — — — — — — — — — G AY62 7292 — G — G T GG G C A Y627293 — — — G G — T GG G C

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 211aaaaaaaaaaaaaaaaaaaaaaaaaaa Intron 2go AY627288 GTACCTAAACCATTTGTGTGTAGTTATTTGTTGTGCTAAT ***** GTTATTTCGAAAAATATGTTTATTT AY62 72 8 9 A TG . — ***** X--G-AA--T--G T AY62 72 90 TG ***** X--G-AA--T- -G T AY62 72 91 A XG ____***** C-- *-TG-_A_ _x*** * * *A AY62 72 92 A XG ATTAT T-- * ~TG--X--X** * * **A AY62 72 93 A XG ATTAT T--*_TG- _X — x** * * **A AY62 72 94 x AT *****__. T-- *-TG--T- -T A AY62 72 9 5 TG ***** - - -X--*_TG- _A_ _x* * * * * *A Taql 130

AAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 2gi Intron 350 :7288 -GAA-* *ATTATAAGGGTTCCTTGTCAGAACTACGGAACCCTAAAAACCTGCTGTAATCTAATAAAAATA ;7289 — GAA — * *ATTATAA — — — G C A :7290 — GAA ~ * * ATTATAA — — G — C — — — — — — A :7291 ***T-*** * a***q q A x ;7292 ***X — ******** A — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — —^ — — — — — — — — — — — — — — — — — ;7293 ** * X — ******** — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — :7294 —AGT — TCACAATAA A C T 17295 * * * X — ******** — — — — — — — — — — — — — — — — — — — — çj — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — —

.^AAAAAAAAAAAAA INTRON AAAAAAAAAAAAAAAAI EIEIIDTNNK420 .7288 ATAAATGCTGCAACCGTTTCGCCTTTAACTTCGACAGATCGAAATAGAAATCATCGACACAAACAACAAA ,7289 A T C : 72 9 0 ---A T C .7291 A T C 7292 A T T 7293 ---C T C 7294 A A C 7295 A T C

4 21V P E PLTEKFNTTVYNVWENATSGDE490 7288 GTGCCTGAGCCGCTCACTGAGAAGTTCAACACGACGGTGTACGTCTGGGAGAATGCCACTATCGGCGACG 7289 -T T 7290 -T T 7291 A c 7292 A c 7293 A c 7294 A T 7295 A T Ksal

Primer OnCadTBRl-R 4 91 V V Q L<<<<<<<<<<<<<<<<<<<<<523 7288 AGGTGGTCCAGCTGTACTCCCACGACCGTGACA 7289 7290 7291 7292 7293 7294 7295 131

Figure 6.5. Alignment of O. nubilalis cadherin CrylA toxin binding region 2 (OnTBR2) genomic DNA sequence GenBank accession nos. AY629584 to AY629592. Primer annealing sites are indicated by arrows showing direction. Region bound by primers

OnCadTBR2-F and OnCadTBR2-R encompasses the PCR product OnTBR2. Identical nucleotides (-) and deletions (*) are with respect to uppermost reference sequence. Introns are overwritten (A) and amino acids encoded in exons are labeled using standard 21-symbol code. Polymorphic Cfol, HaeIII, HpyC H4III, and M s pi restriction endonuc lease sites are underlined and labeled appropriately within the region OnTBR2-s amplified with primers

OnCadTBR2-F and OnCadTBR2-R2.

Primer OnCadTBR2-F !>>>>>>>>>>>>>>>>>>>>> >E GLFVLDQS GSRN V I S L A 70 AY629584 TCTCTTCAACAGACGGCAACAACGAGGGTCTGTTCGTACTGGACCAGTCGCGCAACGTCATCTCCCTTGC AY629585 AY629586 AY629587 AYS29588 T A AA ~ AY62 9589 AY629590 AY629591 AY629592 Cfol Cfol

71QELDREV AT TSYFTLHIAASNSPPDA 140 AY629584 GCAGGAGTTGGACCGCGAGGTGGCCACGTCATACACGCTGCACATCGCGGCGAGCAACTCGCCCGACGCC AY62 95 85 AY629586 AY629587 AY629588 AY629589 AY629590 AY629591 AY629592 Haelll

AAA AAA 141T GIPLQTSILVVTVI N Intron - - 210 AY629584 ACTGGGATCCCTCTGCAGACTTCCATCCTCGTTGTCACGATCAATGTGAGTACATGGATGGAAGTAGCGC 629585 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — - — — — — — — — — — — — — — — — ———————— x* AY629586 G T G A C AY6295 87 G C ~ — G — — — G — C AY6295 8 8 — — — — — — — — — — — — — —Q — — — — — — — — — — — — — — — — —x — — — — — — G — — — — — — — — — — — — — — —G -• — — — — — — — — — — — —l1 Y629589 — — — — — — — — — — — — — — x — — — — — — — — — — — — — — — — —— Q — — — — — — — — — — — — — — —

Primer OnCadTBR2-R2 intron AY629584 ATTTCAAGCTACCTTTCTTCCTGCAAAAGCTCAGTATTTCCAAATAATGGAAACCACCAATGAGCGTCAA AY62 95 8 5 T C C C T ATCGAAACCACCA AY629586 T C G C T ATGGAAACCACCA AY62958 7 T C G C T ATGGAAACCACCA AY629588 T C G C T ATGGAAATC AC CA AY629589 T A C C C ATGGAAACCACCA AY629590 C C C C T ATGGAAACCACCA AY629591 T C G C T GTGGAAACCACCA AY6 29592 T C G T T *************

281 Intron 350 AY629584 TCAGTTTTGTTCTTAACAGTCTCACGCGTTGAGAATTTTTGTACCGCGTTGACGAGTTCTTTGTAACATA AY629585 G T A AY629586 G T A AY6295 87 G T A AY62 95 88 G A G AY629589 A T A AY629590 G T A AY62 95 91 G T A AY62 95 92 A T G

351AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA INTRON AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA420 AY629584 **** TCTATTTACTCGCTGACTACTTTA* AAAATCAGTCAGCTATCATCTAACGGTTCAAATCTTGTGCA AY629585 **** CT T-T TCATCTA T AY629586 * * * * CT T - * ******* Q AY6295 8 7 * * * * CT T - * ******* Q AY629588 **** CT * -T TCATCTA T AY6295 89 **** CT T-T TCATCTA T AY629590 * * * * CT T-T TCATCTA T AY629591 **** AA T-T TCATCTA T AY629592 GTTA CC T-* TCATCTA G

421~V REANPRPIFEQDLYTAGISTLDG 490 AY6 29584 GGTAAGAGAAGCGAACCCGCGCCCAATTTTCGAGCAGGACCTTTACACAGCGGGCATTTCGACGTTGGAC AY629585 C AC AY62 95 86 C AC AY629587 C GC AY629588 C AC AY629589 A AC AY629590 C AC AY629591 C AC AY629592 C AT

Primer OnCadTBR2-R 491S I G R<<<<<<<<<<<<<<<<<<<<<<<<524 AY629584 AGCATTGGCCGGGAATTGCTTACCGTCAGGGTAA AY629585 AY629586 AY629587 AY629588 AY 62 95 89 AY629590 AY629591 AY629592 133

Table 6.1. Mendelian inheritance of cadherin alleles in O. nubilalis pedigrees Ped31

and Ped62. Observed genotypic ration among F2 offspring was compared to Mendelian

expected F2 ratio based on parental genotypes (R15 genotype is R1 and R5 hétérozygote; see

PCR-RFLP alleles on Table 6.1). Significance of observed allele frequency departures from

expected tested by chi-square (%2).

Parental Observed cadherin PCR-RFLP genotype Expected Pedigree genotype ratios among F2 progeny F2 ratio x2 p-value (df) Ped31 (ÎR15ÇR16 TBR1; R11(13/39): R16(12/39): R15( 7/39) 4:4:4:2:1:1 5.92 0.205 (4) RS6( 6/39): R55( 0/39): R66( 1/39) TBR2; CI 1(26/39): C12(14/39): C22( 4/39) 9:6:1 1.07 0.586 (2) Ped62 ^T22$T11 TBR1; T11(10/44): T12(26/44): T22( 8/44) 1:2:1 1.64 0.441 (2)

d,H33$H34 TBR2; H33(26/44): H34(14/44): H44( 4/44) 9:6:1 1.01 0.604 (2) Table 6.2. Mead, Nebraska O. nubilalis frequency of cadherin PCR-RFLP alleles (Table

6.1). Restriction enzyme (RE) fragment sizes in base pair, and alternate amino acid (AA) detected by PCR-RFLP given for each allele. Observed allele frequency (A0) were calculated.

PCR-RFLP RE Fragments AA AQ OnTBRl Rsal Alleles R1 241, 130,61,45,21, 18 0.341 R2 270, 151,61, 18 N866 0.012 R3 275, 151,61, 18 N866 0.085 R4 287, 151,61, 18 N866 0.012 R5 241, 151,61,45, 18 N866 0.244 R6 250, 151,61,45, 18 N866 0.305 OnTBRl Taql Alleles T1 241, 119, 105,29, ... Intron 0.450 T2 224, 119, 105,29, ... Intron 0.431 T3 123, 119, 117, 105,.. Intron 0.060 T4 229, 119, 105,29,... Intron 0.060 OnTBRl Tsp5m Alleles PI Uncut v858 0.085 P2 365,158 yS58 0.220 P3 251, 158, 114 v858 0.384 P4 239, 148, 139 j858 0.311 OnTBR2-s Cfol Alleles CI 174,70 G 1296/gl296 0.455 C2 138,51,36,19 ^12% 0.141 C3 174,51, 19 ^12% 0.327 C4 138, 70, 36 G1296,g 1296 0.077 OnTBR2-s Haelll & //pyCH4IlI Alleles HI 151,93 ^1310 j1339 0.330 H2 177,67 -pl310 yl339 0.304 H3 93, 84, 67 ^1310 y1339 0.357 135

CHAPTER 7. SEQUENCE VARIATION IN TRYPSIN- AND CHYMOTRYPSIN-LIKE cDNAS FROM THE MIDGUT OF OSTRINIA NUBILALIS-. METHODS FOR ALLELIC DIFFERENTIATION OF CANDIDATE BACILLUS THURINGIENSIS RESISTANCE GENES

Brad S. Coates Richard L. Hellmich, and Leslie C. Lewis

Paper submitted to Insect Molecular Biology April 3, 2005

Abstract

Midgut expressed alkaline serine proteases of Lepidoptera function in conversion of Bacillus thuringiensis (Bt) protoxin to active toxin, and reduced levels of transcript T23 is associated with Ostrinia nubilalis resistance to Dipel® Bt formulations. Three groups of trypsin-

(OnT25, OnT23, and OnT3) and two chymotrypsin-like (OnCl and OnC2) cDNAs were isolated from O. nubilalis midgut tissue. Intraspecific groupings are based on cDNA similarity and peptide phylogeny. Derived serine proteases showed a catalytic triad (His,

Asp, and Ser; except transcript OnT23a), three substrate specificity-determining residues, and three paired disulfide bonds. RT-PCR indicated all transcripts are expressed in the midgut. Mendelian-inherited genomic markers for loci OnT23, OnT3 and OnCl will be useful for association of alleles with bioassayed Bt toxin resistance phenotypes.

Keywords

Ostrinia nubilalis, serine proteases, Bacillus thuringiensis resistance 136

Introduction

Transgenic crops that express insecticidal toxin proteins derived from Bacillus thuringiensis (Bt) reduce or eliminate feeding by pest insects (Crickmore et al. 1998; Walker et al. 2000). The CrylAb gene was incorporated into corn (Koziel et al. 1993) and other crop plant germplasm (Frutos et al. 1999). Commercial corn hybrids expressing CrylAb toxin have been available since 1996 (Rice and Pilcher, 1998) and in 2004 were planted on 29% of

U.S. com ground (22.9 million ha; USDA-NASS, 2004). Transgenic CrylAb coding sequences have been truncated (3' and 5') and codons altered from the native B. thuringiensis subsp. kurstaki gene (Vaeck et al. 1998), which might affect crystallization or proteolytic activation requirements by endogenous proteases in susceptible insect midguts (Schnept et al.

1998; Rukmini et al. 2000).

Moderate Ostrinia nubilalis resistance to Cry toxins was observed after laboratory selection (Bolin et al. 1999; Huang et al. 1999b; Chaufaux et al. 2001), and frequency estimates of resistant phenotypes in natural populations are < 9.2 x 10~4 (Andow et al. 1998;

Andow et al. 2000; Bourguet et al. 2003). Reduced expression of a major trypsin protease in midgut tissue was shown for Plodia interpunctella phenotypes resistant to CRYlAb (Oppert et al. 1996; Zhu et al. 2000). Midgut extracts from an O. nubilalis strain, KS-SC reared on

Dipel® Bt formulation, had reduced trypsin-like protease activity and 35% decreased capacity to hydrolyze CRYlAb protoxin compared to susceptible controls (Huang et al.

1999a). Li et al. (2004) showed a reduced rate of toxin activation in some O. nubilalis strains was correlated with decreased trypsin-like protease activity. Recently, Li et al. (2005) demonstrated a 2.7 to 3.8-fold reduction in trypsin T23 transcript level in Dipel® resistant compared to susceptible individuals. 137

Families of serine protease genes are expressed in the epithelial cells of insect midguts (Peterson et al. 1994; Brown et al. 1997). Trypsin-like proteases cleave following highly basic residues (Arg and Lys), and are determinants of B. thuriengensis protoxin activation (Haider and Eller, 1989; Oppert et al. 1997; Rukmini et al. 2000; Li et al. 2004 and

2005). Chymotrypsin-like proteases cleave following aromatic residues (Trp, Tyr and Phe), and may degrade active (trypsinized) Cry toxins, rendering them nontoxic to susceptible insects (Yamagiwa et al. 1999; Miranda et al. 2001). Culexpipiens chymotrypsins were implicated in breakdown of active Cry4A toxins (Yamagiwa et al. 1999). Native CRYlAb tolerance of Spodoptera frugiptera might lie with its high active toxin degradation rate compared to more susceptible Manduca sexta larvae (Miranda et al. 2001). Although not implicating chymotrypsins, Keller et al. (1996) also showed increased rates of CRY1C toxin degradation in late instar Spodoptera litoralis was due to enhanced serine protease activities.

Midgut serine proteases are part of a CRYlAb activation pathway, and represent a

Dipel® Bt toxin formulation resistance mechanism in O. nubilalis (Huang et al. 1999a; Li et al. 2004 and 2005). An O. nubilalis trypsin-like enzyme was isolated by fractionation of midgut juices (Bemardi et al. 1996), midgut protease activity properties characterized

(Houseman et al. 1989), and individual transcripts isolated (Li et al. 2005), yet intraspecific variation and molecular markers for these O. nubilalis genes have not been described. We report characterization of 18 unique trypsin- and chymotrypsin-like transcripts expressed in midgut of O. nubilalis. Molecular markers for genomic- and expression-level analysis were developed, and may be used in linkage mapping or studies to associate co-segregation of alleles with bioassayed O. nubilalis Bt toxin resistance phenotypes. 138

Experimental Procedures

RNA sample tissue

Bivoltine Z-pheromone strain O. nubilalis adults were field collected at the Iowa

State University Uthe Farm and maintained in culture at the USDA-ARS Corn Insects and

Crop Genetics Research Unit (CICGRU) facilities in Ames, IA for 3 to 5 generations.

Larvae were reared on a semi-meridic diet (Guthrie 1987), midguts dissected from six 5th

instars, samples pooled, and ground in liquid nitrogen with a mortar and pestle. Additionally, fat body, midgut, and head were dissected from two 5th instars, tissues kept separate, and

ground to powder in liquid nitrogen. RNeasy extraction kits (Qiagen) were used according to

manufacturer instructions to obtain total cellular RNA from 10 mg of ground O. nubilalis

tissues. The extracts were quantified by absorption at 260 nm (A260), diluted to 50 ng/jal with

nuclease free (0.1% DEPC treated) water, and used immediately.

Complementary DNA (cDNA) synthesis and cloning

First strand cDNA synthesis used 0.5 |iM of a poly(T) adapter (PT-AD; 5 '-GGT GTA

ATA CGA CGG CCT GGA ATT CTT TTT TTT TTT TTT TTT T-3 ), 2 \ig of total RNA,

40 U of AMY reverse transcriptase (Promega, Madison, WI) in a 20 pi reaction that was

incubated at 42°C for 1 h on a PTC-100 thermocycler (MJ Research, Watertown, MA).

Subsequent PCR used 2 p,l first strand cDNA as template, 5 pmol degenerate primer 5 -CAG

GGT GAC TCY GGC GGY C-3 ' (corresponding to the conserved QGDSGGP peptide sequence; Mazumdar-Leighton et al. 2000), 5 pmol poly(T) adapter core primer 1 (PT-Adcl;

5 -GTG TAA TAC GAC GGC CTG G-3 '), 0.9 U Tli polymerase (Promega), 0.1 U Taq 139 polymerase (Promega), 50 pM dNTPs, 1.5 mM MgCh, and 2.5 pi of Thermopolymerase

Buffer (Promega) in a 25 pi reaction. A PTC-100 thermocycler (MJ Research) performed 35 cycles of 95°C for 30s, 60°C for 20s, and 72°C for 30s, followed by 72°C for 10 m.

Amplification success was confirmed and fragment sizes estimated by electrophoresis of 10 pi PCR product on a 10 cm 1.5% agarose IX TBE (Tris-Borate-EDTA, pH 8.0) gel containing 0.5 pg/ml ethidium bromide. PCR products were ligated using the pGEM-T easy cloning system (Promega), incubated overnight at 4 °C, and used to transform 80 pi of E. coli

SURE (Stratagene) by electroporation. Sixteen insert positive plasmids containing 3' RACE products were identified, and each sequenced using 10 pi DTCS Quickstart DNA sequencing reactions (Beckman-Coulter) according to manufacturer instructions with 1.6 pmol T7 primer. Primer extension products were purified by ethanol precipitation, suspended in 40 pi deionized formamide, and separated on a CEQ 8000 Genetic Analysis System (Beckman-

Coulter) with method LFR-1 (denature: 90°C for 120 sec; inject: 2.0 kV for 15 sec; and separated: 4.2 kV for 85 min in a 50°C capillary). Sequence data was used to design transcript-specific reverse primers; OnT25-5pR (5'-ATC ATC AGC ATC CGT AAC GTG-

3 '), OnT23-5pR (5 -TTG ACA CCA GGG AAG AAA GCG-3 '), OnT3-5pR (5 -TAT TAA

AC A GCA GTA GCC GCG-3 '), OnCl-5pR (5 -GAT GTT GTT TAT AGG TGC TGG TTG

A-3 '), and OnC2-5pR (5 '-GCA ATC AAT CAT TGG CTG CTC AA-3 ').

First Choice RNA Ligase-Mediated (RLM) RACE kits (Ambion) were used to recover 5' cDNA ends for each transcript in accordance with manufacturer instructions.

RLM-RACE template was generated in reactions that used 1 pg of total RNA, and transcript- specific reverse transcriptase (RT)-PCR products generated separately from primers located 140 in the 3'-UTR in conjunction with RLM-RACE 5' outer primer. Subsequent 25 gl hot start

PCR used 1.0 U Tli polymerase (Promega), 200 |AM dNTPs, 1.5 mM MgCl2, and 2.5 |_il of 10 x thermal polymerase buffer (Promega), 1 (j.1 RLM mRNA template, 5 pmol of transcript specific reverse primer and 1 (J.1 RLM-RACE 5' adapter outer primer (Ambion; 5 -GCT GAT

GGC GATG AAT GAA CAC-3'). A PTC-100 thermocycler (MJ Research) cycled 40 times at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 m. RT-PCR products were ligated and used to transform the E. coli SURE host strain. Forty clones carrying plasmid inserts containing 5'

RACE products were identified (9 OnT25, 1 OnT23, 10 OnT3, 10 OnCl, and 10 OnC2), and

DNA sequence was obtained as previously described.

Trypsin- and chymotrypsin-like protease phylogeny

Twenty six lepidopteran trypsin and chymotrypsin peptide sequences were obtained from GenBank, and used to construct a 318 amino acid consensus alignment after inclusion of derived amino acid sequence from 18 unique cDNAs encountered in the present study.

All subsequent manipulations use the PHYLIP software package (Felsenstein 1989). One thousand bootstrap resampling steps were produced by the SeqBoot program, parsimony trees were generated using ProtPars, a strict consensus tree was estimated from all possible phytogenies with CONSENSE, and was viewed using TreeView (Page 1996).

Trypsin- and chymotrypsin-like protease expression

Total RNA extracted from fat body, midgut, and head of 5th instar O. nubilalis was subjected to RT-PCR analysis. Individual first strand cDNA synthesis reactions used 250 ng 141 total RNA template, 10 pmol of reverse primer (Table 7.1) or |3-actin-R (5 '-GAC AAC GGC

TCC GGT AT G T-3 controls only), 2.5 U Tth polymerase (Promega), 100 jaM dNTPs, 2.5 mM MnCl2, and 2.0 JJ.1 of 10X Tth reverse transcriptase buffer (Promega) in a 10 pl reaction.

A PTC-100 thermocycler (MJ Research) performed a primer extension cycle of 85 °C for 1 m, 50 or 56 °C for 1 m, and 72 °C for 20 m. A 4.0 pi aliquot of 1st strand cDNA synthesis product was mixed with 1.6 j_il chelate buffer (Promega), 10 pmol of primer forward, or |3- actin-F (5 -CCT TCG TAG ATA GGG ACG GT-3 controls only), and MgCl2 concentration adjusted to 2.0 mM in a 20 pi final volume. PCR reactions were carried out on a PTC-100 thermocycler (MJ Research) using 40 cycles of 95 °C for 30 s, 50 or 56 °C for 30 s (Table 7.1), and 72 °C for 1 m. RT-PCR products (20 pi) were separated on a 10 cm 1.0 % agarose IX tris-borate EDTA gel containing 0.5 pg/ml ethidium bromide, and digital images taken under UV illumination on a BioRad ChemiDoc System (BioRad, Hercules, CA).

Genomic marker development

Mendelian inheritance of PCR-RFLP markers was evaluated by observing allele segregation in pedigrees PedlOb and Ped24a. Two paired matings of O. nubilalis adults from the USDA-ARS, CICGRU laboratory colony established Fi families. The F, progeny were sib-mated and resultant F2 larvae reared on a semi-meridic diet (Guthrie 1987). DNA was extracted according to Coates and Hellmich (2003), and samples stored at - 20 °C prior to use.

Original cDNA sequences were aligned using AlignX (Informax, San Francisco, CA).

Polymorphic restriction endonuclease cleavage sites were identified. Single nucleotide 142 polymorphisms (SNPs) were detected by PCR-RFLP, with amplification of OnT23, OnCl,

and OnC2 gene fragments using primers in Table 7.1. OnT25 and OnT3 used different reverse primers, OnT25-R2 (5-GAT GTC AAG GGC GGT TCG GA-3') and OnT3 (5'-

GTA CAC GAA GGGGGT GAC CA-3 '), respectively. PCR reactions (10 pi) used 0.25 U

Taq polymerase (Promega), 200 pM dNTPs, 1.5 mM MgCb, and 1.0 pi of 10 x thermal polymerase buffer (Promega), 100 ng of genomic DNA, and 2 pmol of each forward and reverse primer. The OnT25 genome fragment was digested with Rsal or Haelll. OnT23,

OnT3, and OnCl were digested with Haelll, and OnC2 with Cfol. All endonucleases were purchased from Promega. PCR-RFLP reactions included 5.0 pi of appropriate PCR product,

2.5 pi lOx Buffer, 0.1 mg/pl BSA, and 0.25 U of enzyme in 25 pi. Reactions were incubated at 37 °C or 60 °C (TaqT) for 14 h. Entire PCR-RFLP reactions were loaded onto 10 cm 2 %

IX TBE agarose gels containing 0.5 pg/ml ethidium bromide. Samples were separated at

100 V for 1 h, and images captured under UV illumination on a ChemiDoc System (BioRad).

Fragment sizes are estimated using Gel-Pro Analyzer (Media Cybernetics) by comparison to a pGEM5Zf(+) Mspl digest ladder (765, 489, 404, 342, 242, 191, 147, and 110 bp). Chi-

2 square tests (% ) measured goodness-of-fit between observed allelic distributions among F2 and Mendelian ratios expected from parental genotypes.

Results and Discussion

Complementary DNA (cDNA) sequences

Eighteen unique alkaline serine protease-encoding sequences (including 2 partial sequences; Fig. 7.1) were observed from 40 clones containing cDNAs from bivoltine Z- 143 pheromone strain 5th instar O. nubilalis reared on semi-meridic diet (Guthrie, 1987). All sequences were submitted to GenBank (Table 7.2). BlastX and Protein Database (PDB) searches (Gish and State, 1993, Rost, 1996) showed high similarity of 11 O. nubilalis proteins to trypsin-like peptides and 7 to chymotrypsin-like peptides from other Lepidoptera

(GenBank accessions incorporated into Fig. 7.2). Five trypsin-like cDNAs showed 98 to

Sixteen complete cDNA (omitting 2 partial sequences OnCl a and OnC2c; Fig. 7.1) sequences ranged from 821 to 923 nt, with the shortest transcript having a block deletion between consensus codons 44 to 73 (OnT23a; Fig. 7.1 A). A truncated 156 residue trypsin- like protease, OnT3e, was predicted due to a premature stop codon (TGA), and confirmed by duplicate and overlapping sequencing runs. Two partial chymotrypsin-like cDNA sequences of 677 and 859 nt also were obtained that respectively were missing N- and C-terminal coding regions (OnCl a and OnC2c; Table 7.2). OnCl a and OnC2c may represent cloning artifacts (Brown et al. 1997), but were retained within the alignment due to polymorphism they contain. Omitting two partial cDNA sequences and the truncation mutant, 15 cDNA had open reading frames that ranged from 231 to 291 codons (Fig. 7.1 A, B).

Pairwise sequence similarities among O. nubilalis cDNA sequences were generated from a 1030 nt long consensus cDNA alignment and showed 5 groups of serine proteases may exist (Table 7.3); three O. nubilalis trypsin-like (OnT25, OnT23, and OnT3) and two 144 chymotrypsin-like transcript groups (OnCl and OnC2). Five OnT25 transcripts shared >

OnT23 cDNA showed 65% similarity with OnT25 and 71 to 73% similarity to the OnT3 class of trypsin cDNAs, but shared 83% cDNA similarity to the O. nubilalis trypsin T23 that was implicated in Dipel® resistance traits (Li et al. 2005; Table 7.3). Similarly, two chymotrypsin-like transcripts (OnCl and OnC2) showed high intragroup similarities, > 85% among OnCl and > 75% among OnC2 groups. Because cDNA was isolated from a pooled sample containing midgut tissue from multiple individuals, group OnT25, OnT23, OnT3,

OnCl, and OnC2 each may contain different alleles from the same locus. Alternatively, each group might have representatives of different highly related genes (different loci) that are in early phase of differentiation (Zhu et al. 2005). This issue was addressed using Mendelian inheritance data (discussed later). Complementary DNAs resembling the 1477 nt T2 transcript (Li et al. 2005) were not observed in this study, and Li et al. (2005) did not describe OnT3-like cDNAs. Reason for difference in transcripts isolated between the two studies might be variance in transcript levels due to different genetic backgrounds of colonies used, mutation or random genetic drift in the highly inbred KS-SC colony and isoline (44 and

47 generations in culture, respectively), or difference in cDNA isolation methods used.

Encoded peptides are translated as inactive zymogens that require enzymatic or autocatalytic activation (Graf et al. 1998). Thus all 16 derived alkaline serine proteases are zymogens, trypsinogen or chymotrypsinogen (obtained from full-length open reading frames only) that are excreted into the gut lumen prior to activation. Cytoplasmic localization and excretion of all derived peptides was predicted by PSORTII (Probability >94.1; 145 http://www.psort.org/) using methods by Reinhardt and Hubbard (1998). Protease activation involves removal of N-terminal leader peptide regions. Previously, Bemardi et al. (1996) purified a 24.65 kDa trypsin-like protease with 20 residue N-terminal sequence IVGGS[XI5], which starts at position 24 of the derived ONT25a peptide. The Bemardi et al. (1996) data suggest O. nubilalis ONT25 and ONT23 trypsinogens are cleaved preceding a conserved

IVGG signal that releases a 23 residue leader peptide rich in positively charged amino acids

(Peterson et al. 1994; Brown et al. 1997; Zhu et al. 2000; Li et al. 2005). Analogously, cleavage preceding an IIGG peptide sequence may result in a mature active ONT3 (Fig. 7.1

A), releasing a 22-residue leader peptide. Ostrinia nubilalis chymotrypsinogen leader peptides are longer compared to trypsinogen counterparts. Mature ONC1 and ONC2 chymotrypsin peptides might be respectively formed by cleavage prior to IVGG and IWGG sequences (Fig. 7.1 B), which was analogous to predictions from Manduca sexta (Peterson et al. 1994), and A. ipsilon and H. zea (Mazumdar-Leighton and Broadway 2001).

Following cleavage, mature active enzymes are predicted to have molecular weights of 24.69 to 24.81 (ONT25), 22.67 or 24.79 (ONT23), 25.89 to 26.43 (ONT3), 24.71 or 24.84

(ONC1), and 23.66 kDa (ONC2; Table 7.2). Li et al. (2004) showed 3 soluble trypsin-like peptides (240, 85, and 34 kDa) and two soluble chymotrypsin-like peptides (53 and 29 kDa) from O. nubilalis midgut, suggesting our cDNA sampling is representative of the low molecular weight range of enzymes. Differences in estimated molecular weights between our study and larger molecular weights from Li et al. (2004) might be influenced by posttranslational modifications that retard peptide migration during electrophoresis.

Zymogen analysis with CRYlAb as substrate refined estimates of toxin degrading enzyme mass to 55, 38, 28, and 23 kDa (Li et al. 2004). 146

Catalytically active residues of serine proteases are referred to as the catalytic triad

(Botos et al. 2000), and substrate-specificity determined by residues near the carboxy-

terminus (Krem et al. 1999). Conserved trypsin-like protease residues His69, Asp138, and Ser

221 (catalytic triad), three paired Cys that may form disulfide bonds, and 3 substrate specificity determining residues, Asp215, Gly238, and Gly248 were encoded by OnT25, OnT3 cDNAs, and trypsin T23 (Li et al. 2005; Fig. 7.1 A).

A single transcript, OnT23a, had an internal deletion that omitted the codon for His70, suggesting a nonfunctional enzyme (Fig. 7.1 A). Our OnT23a transcript cDNA was 83% similar to that of O. nubilalis trypsin T23 (AY513650) that did encoded a catalytic His70.

The observed RT-PCR product length using OnT23-F and OnT23-R primers did not correspond to size predicted from the OnT23a transcript (226 bp), but was similar to that

from the T23 transcript (313 bp). Experimental results suggest amino acids 45 to 73, including His70, likely was present in expressed OnT23 alleles sampled in this study (Fig.

7.3). The OnT2a sequence may be a sequencing artifact, rare mutant, or unexpressed allele variation.

Chymotrypsin-like peptides ONC1 and ONC2 showed invariable His98, Asp145, and

Ser242 (catalytic triad), and six Cys for disulfide bond formation (T; Fig. 7.1 B). First residue of the chymotrypsin substrate specificity-determining domain was variable (Gly/Ser238), but remaining Gly263 and Gly274 were fixed among O. nubilalis sequences. All members of cDNA group OnCl encoded Gly238, while all ONC2 chymotrypsins are predicted to have

Ser238. Analogous variation between Gly/Ser was observed between A. ipsilon and H. zea

(Mazumdar-Leighton and Broadway 2001), and might be involved in substrate specificity 147 differences (Botos et al. 2000). No O. nubilalis serine protease group showed elastase-like valine or threonine in the latter specificity determining residues.

Trypsin- and chymotrypsin-like protease phylogeny

Trypsins and chymotrypsins may have differentiated from a single common ancestral gene (Neurath et al. 1967), and the following phylogeny was used to derive inter- and intraspecific relationship among serine proteases. Derived peptide sequences derived from

18 variant O. nubilalis serine-like protease cDNAs were placed into a peptide phylogeny with 26 midgut protease-like peptides from Lepidoptera (21 trypsin-like and 6 chymotrypsin- like). Two lineages (trypsin- and chymotrypsin-like) were indicated from parsimony tree construction, with the separation strongly supported (993 of 1000 bootstrap iterations; Fig.

7.2). Bootstrap branch support was >71.3% of 1000 bootstraps for all but two internal nodes.

GenBank accession HAY12270 was separated from other H. armigera trypsins in 418 of

1000 random trees and the node separating Galleria mellonella (AY04081) from P. interpunctella trypsins was supported by 420 of 1000 bootstrap iterations, suggesting indeterminate genealogical relationships at those nodes. Parsimony analysis clustered 13 O. nubilalis peptides ONT25, ONT23, and ONT3 into three phylogenetic groups nested within trypsin-like proteases. ONT25 derived peptides may show common ancestry to M. sexta trypsins (Lepidoptera: Sphingidae; MOTTPPA, B, and C). ONT23 and ONT3 peptides grouped separate from the ONT25 groups, but show a common ancestral gene. The phylogeny indicated ONT23 (and AY513650) were more highly related to ONT3 compared to ONT1 group peptides, which supported conclusions drawn from cDNA sequence similarities (71 to 75%; Table 7.3). Trypsins from the pyralid P. interpunctella did not group 148 near those from O. nubilalis, suggesting high levels of peptide variation outside of functional residues.

Derived peptides ONC1 and ONC2 were separated from heliothine (H. zea and A. ipsilon) chymotrypsins by 100% of reconstructed trees, and ONC1 and ONC2 peptides comprised two differentiated groups (1000 of 1000 bootstraps). The O. nubilalis chymotrypsin-like proteases may form a unique clade or lineage. Alternatively, the few number of isolated lepidopteran chymotrypsin genes might contribute to lack of close phylogenetic relatedness. Incorporation of additional yet uncharacterized chymotrypsin sequences are required to fully resolve the phylogeny.

Trypsin- and chymotrypsin-like protease transcription

Reverse transcriptase (RT)-PCR indicated O. nubilalis trypsin and chymotrypsin-like transcripts for OnT25, OnT23, OnT3, OnCl, and OnC2 are present in midgut tissue (Fig.

7.3). Members of serine protease gene families are expressed in midgut epithelial cells and function as peptide digesting enzymes (Peterson et al. 1994; Brown et al. 1997). The OnT25,

OnT23, OnT3, and OnC2 transcripts also were present in total RNA extracted from O. nubilalis head tissues, and may indicate salivary gland expression (note: OnT23 head expression is very low; Fig. 7.3). Serine proteases in salivary juices may function in digestion prior to entry into the alimentary canal (Cohen, 1998). The Hessian fly expresses two chymotrypsins, MDP1 and MDP2, and two trypsins, MDP3 and MDP4, in gut and salivary tissue (Zhu et al. 2005), whereas a third trypsin, MDP5, was specifically expressed in the gut. The trypsin transcript HaPl from H. armigera also was expressed only by midgut tissue. Only OnCl was specifically expressed by the midgut, but OnT23 only showed low 149 expression in head tissue compared to midgut. Additionally, RT-PCR detected OnT25- and

OnC2-like transcripts in O. nubilalis fat body tissues (Fig. 7.3), suggesting lepidopteran serine protease genes have different degrees of tissue specificity. The P-actin control showed equal RT-PCR amplified band intensities for all tissue samples, except the second replicate of total RNA derived from fat body tissue. Decreased band intensity likely was due to experimental error (reaction or gel loading) as opposed to transcript level, since products

OnT25, OnT23, OnT3, OnCl, and OnC2 were of equivalent intensity as the first replicate of the fat body tissue sample. All RT-PCR product lengths were as predicted from cDNA sequence (Table 7.1), except OnT23a (explanation offered previously).

Genomic markers

Polymorphic genomic markers were developed for the O. nubilalis genes OnT23,

OnT3, OnCl, and OnC2, and used to investigate Mendelian inheritance pattern and show single locus specificity. Intraspecific alignment of O. nubilalis protease cDNAs identified

17, 14, 22, 32, and 10 single nucleotide polymorphisms (SNPs) among OnT25, OnT23,

OnT3, OnCl, and OnC2 cDNAs, respectively (alignments not shown). Single genomic DNA fragments containing respective trypsin or chymotrypsin gene were PCR amplified using same primers as in RT-PCR (Table 7.1). Some SNPs within amplified regions were detected by PCR-RFLP assays. Polymorphic OnT23 and OnT3 fragment patterns resulted when digested with HaeIII, and similarly OnCl with HaeIII or Hinfl, and OnC2 with Cfol. No variation was observed among alleles at the OnT25 locus, and was not used in subsequent pedigree analysis. Polymorphism of PCR fragments may result from the co-amplification of > 1 loci or represent allelic variation at a single locus, and the latter can be established by observation of Mendelian inheritance patterns.

OnT3 HaeIII and OnC2 Cfol markers were polymorphic within pedigree PedlOB, and

OnT23, OnT3, and OnCl Haelll variation was observed within Ped24a. Parents and F2 offspring genotypes were determined at all polymorphic loci, and data used to test Mendelian

2 inheritance. Chi-square tests (% ) determined that the allelic distribution among F2 individuals did not deviate significantly from expected Mendelian ratios determined from parental genotypes, except for alleles of the OnC2 locus (Table 7.4). Deviation likely resulted from segregation of 2 null alleles at the locus, co-amplification of alleles from > 1 locus, or non-random mating among Fi progeny. Therefore, the O. nubilalis OnT23 HaeIII,

OnT3 Haelll, and OnCl Haelll PCR-RFLP defined alleles are known inherited in a

Mendelian manor in the two pedigrees tested (Table 7.4). This suggests OnT3, OnCl, and the Dipel® resistance associated OnT23 genes are locus-specific PCR products that are appropriate for genomic linkage mapping studies.

Molecular screening is increasingly being incorporated into resistance diagnostics

(Brogdon and McAllister 1998), and comparative studies showed association of three cadherin alleles (rl, r2, and r3) with P. gossypiella Bt resistance traits (Morin et al. 2003).

Assays also were developed to screen O. nubilalis cadherin allele (Coates et al. 2005).

Codominant genetic markers used must show Mendelian inheritance and be devoid of non-

PCR amplifying null alleles for use in phenotypic linkage or association mapping studies

(Pemberton et al. 1995). The T23 Haelll marker did show Mendelian inheritance pattern

(Table 7.4). The marker will be applied in future studies involving inheritance of Bt toxin 151 resistance phenotypes, and used in linkage mapping or studies to associate co-segregation of alleles with bioassayed O. nubilalis Bt toxin resistance phenotypes.

Acknowledgements

This research was a joint contribution from the USDA, Agricultural Research Service and the

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Figure 7.1. Trypsinogen and chymotrypsinogen-like protease sequences derived from

Ostrinia nubilalis midgut cDNAs. A) Alignment of 11 trypsinogen-like protease sequences.

Active site residues His70, Asp138, and Ser221 (highlighted), three disulfide bonds comprised on Cys residues (T), and substrate specificity-determining residues Asp215, Gly238, and Gly248

(double underlined). B) Alignment of 7 chymotrypsinogen-like protease sequences. Active site residues His98, Asp145, and Ser 248, disulfide bonds comprised of three paired Cys residues, and substrate specificity determining residues Gly/Ser238, Gly263, and Gly274. For both alignments, each • indicates an identical residue to its uppermost sequence within each serine protease group, - indicates an alignment gap, and * is a residue deletion. Sequences

T25 (AY513649) and T23 (AY513650) are from Li et al. (2005). 157

A) Ostrinia nubilalis midgut trypsin-like serine proteases.

Leader peptide Cleavage H OnT25a ( 1 ) MRTFIVLLLGLAAVSAYPKNIORki+itySVTSINOYPKMAST.T.FSWC;- TTGHRQACGGT I LNNRS I LTAAH OnT2 5b OnT2 5c OnT2 5d •A- OnT2 5e • I - • • • -S A T25 • I - - - S A OnT23a M-VRLVLLTLALFAGCCYA-APRlnWeidOFTTTNFlYPSTVOVFIFT, T23 • - - • GIFSQAWSQSCAANILSSRYVLSAAH OnT3a MAKFLVLAVALLAVSSCSA-FHRqgggjQEATIEQYPSIVQVEFSNLLGTTWSQSCAANILNVLYVLSAAH OnT3b OnT3c 0nT3d 0nT3e D OnT2 5a (71) CT--IGDAPARWRTRVGSTNANSGGTELATISIINHPNYNGWT-IDNDVSIIRTALNIPIGSSTIQAGRI OnT25b • • QT - V OnT25c • • QT - V OnT 25d QT V • V • • • OnT2 5e • • QT - V T2 5 • • QT - V OnT2 3a GIFYSPSLRRIRAGTTFRNSGGFTRNVANEYNHPTYGLLG-ADGDITWRLAEPLEY-NPWQAGYI T23 CFAG - OnT3a CFEGTTYSPRLRRIRSGTATRNNGGAINYIEREINHPEYRVAARFDADITWRLVTPFVY-SLQVQQGVI OnT3b K - OnT3c • • • -A - OnT3d • • • A - OnT3e • • • *A - t

OnT25a(141) AGANYNLADNQIVWATGWGRTS SGG- -PASEQLRHVQIWTINQAICRQRYATVG--DTITDNMLCSGWLD OnT25b P-D- - -V OnT25c - • V OnT25d - V OnT2 5e - -V -- T25 - -V R OnT23a ANPNTVFPDNQPVIHAGWGHTQFGG- -HPSDVLRHVTIFTINHAICRDRYATLG--WHVTENMICAGLLD T23 I - - P OnT3a VYQDATIPDGLEWHAGWGTTWGDSSTMSPVLLDTIIYTVNNNLCRERYLTLPNPGFVTANMICAGLLD 0nT3b A OnT3c A OnT3d OnT3e .************************************ ***************

OnT2 5a (211) VGGRgQCQGDSGGPLYHNGWVGVCSWgRGCAQAFFPgVNARVSRFTSWIQNNA AY953059 OnT2 5b • • • 4 ^ AY953060 OnT2 5c • • • — ^ ^ AY953 061 OnT2 5d • • • • • -G ^ ^ AY953062 OnT2 5e • • • — 4 4 AY953063 T2 5 • • • — 4 ^ AY513649 OnT23a VGGRgACQGDSGGPLYHGSILVGWSWgHGCANETFPgVSTNVASYTNWIRGYCCLI AY953068 T2 3 T E • • • • A A • • P • • S • • VSVAV — AY513 650 OnT3a VGGRgACQGDSGGPLYYRNILVGWSWgHGCANETFPgVSTNVASYTNWIRGYCCLI AY953064 OnT3b • • • - ^ ^ AY953065 OnT3c - R — ^ ^ AY953066 OnT3d • • • — ^ 4 AV** AY953067 OnT3e ********************************************************* j^Y953 052 T T 158

B) Ostrinia nubilalis midgut chymotrypsin-like serine proteases.

Leader peptide Cleavage OnClb (1) MKFLWLLAVASLAHGKVV*PDNHTAFGYLKNSIVEAEKIRVREEQYLQQORliIMoPANTlGOVPFOAGM le T X'** * * A * * * * • • • "'G ••••L OnCld T I A BBT ' G L OnClaB * A ******************* OnC2a MKSAVWFLLWAAAAA--ERLQPNTRYHETEGIPRMQEIQRLEEGTDFDGGRgjpS!g!OAVGAGTnPHTlrîC;Tl OnC2b L gggQ OnC2cB **************** — ****************************************************

H98 OnClb (71) LINIIGFEGRAVCGAVLISADRLVSAAHCWSDGQHQAWRVEWLGSVTLFTGGNRQFTSVFINHPSWFPL OnClC ' LDV GS • • • * N * VI N • V I OnCld ' LDV GS * • • * N • VI N * V I la *******************• LV • "\j j OnC2a VITLTTG-QLSICGSSLISNTRSVTAAHCWRTNTFQARQFTWWGSNALMSGGTRWTTNVWHPQYNAN OnC2b - S OnC2cB *********** G t t

D145 OnClb (141) LVRNDIGVIYLPTSVSFSNTIAPVSLPQGAELQEDFAGASAIASGFGLTVDGGSISSNQLLSQVRLNVLS • • • • • ^ T * * S * 1d ^ T*"S • • • • E • T F • • • ^ • « T * * S p i « « • . « • « 'j1 p • . » t OnC2a NLNNDVAVIRHN-SVAFNNVINRIALATG SNSFAGTWAVAAGYGRNGDGAGSS -NPGKSQANLLVIT OnC2b - - Qj^Q2 c • • • — — — ••••2""" — ••••F-*

G/S^' S"" G'" G"' OnClb (211) NSECRLGFPLILQDSNICTSGIGGVgTCSGDSGGPLYITRgNGNVLIGVTSFglALGCQVNFPAAYARVT OnClc • -V- -F ^ ^ OnCld ^ j, ^ onci . . v. . p ********************* OnC2a NDVCRQTFGNTIVASTLCVSTAHGSgTCPGDSGGPLAVGSSNNRQLIGITSFg-TQWCARGFPAGFARVT OnC2b 4 ^ 4- OnC2cB ^ 4 t t T t

GenBank OnClb (281) SFMPFINQHL AY953053 OnClc AY953054 OnCld AY953055 OnClaB AY953069 OnC2a SFASWLSSQ* AY953 056 OnC2b * AY953057 OnC2cB * AY953058

B: partial sequence. 159

Figure 7. 2. Ledipteran tripsinogen and chymotrypsinogen peptide sequence phylogeny using parsimony and incorporating 1000 bootstrap iterations. GenBank accessions for 18 0. nubilalis serine protease peptides (ONT25, OnT23, ONT3, ONCI, and ONC2) are indicated.

Additional peptides are from Agrotis ipsilon (AF233728, AF233729, AF233730, AF261970,

AF261971), Helicoverpa zea (AF233731, AF233732, AF233733, AF261980), Galleria mellonella (AY04081), Helicoverpa armigera (HAY12269, HAY12270, HAY12271,

HAY12275, HAY12276, HAY12277, HAY12283), Manduca sexta (MOTTRYPA,

MOTTRYPB, MOTTRYPC), and Plodia interpunctella (AF173495, AF173496, AF064525,

AF064526, AF173497, AF173498). 160

NP523518 D. melanogaster HAY12270 HAY12283 HAY12271 HAY12269 H. armigera HAY12276 4F= HAY12275 975 HT HAY12277 L-lïooo AF261980I H. zea AY04081 |G. mellonella AF173495 AF173496 P. 1000 AF064525 interpunctella ——[Tool AF064526 AF261971 41000 A. ipsilon AF261970 AF173497 P. <1000 AF173498 interpunctella AY953068 O. nubilalis 993 AY613540 (ONT23) AY9530G5 AY953066 O. nubilalis AY953067 (ONT3) AY953064 AY953052 AY953059 AY953060 O. nubilalis AY9530G1 (ONT25) AY953062 AY953063 MOTTRPC MOTTRPA M. sexta MOTTRPB

1000 AF233730 AF233728 A. ipsilon AF233729 AF233732 AF233731 H. zea AF233733 AY953069 AY953053 O. nubilalis AY953054 (ONC1) AY953055 AY953056 O. nubilalis AY953057 (ONC2) AY953058 161

Figure 7.3. Trypsin- (OnT25, OnT23, and OnT3) and chymotrypsin-like (OnCl and

OnC2) transcript reverse transcriptase (RT)-PCR detection among tissues. Total RNA samples: m = midgut; h = head; fb = fat body. L = pGEM5Zf(+) Mspl digest (765, 489, 404,

342, 242, 191, 147, and 110 bp).

OnT25 OnT23 L m mh h fb fb fbfbmmhh L

—— - 765 bp

ZZ "342 bp

OnT3 L m m h h fb fb L h h fb fb L

Bg Aw. ^4 ^ ; w» 489 bp

OnC1 OnC2 mmhhfbfbLmmhhfbfbL . nmijg —. 765 bp g ' — ^-489 bp

^ Table 7.1. Ostrinia nubilalis trypsin- and chymotrypsin-like serine protease gene primers used for reverse transcriptase (RT)-PCR and genomic DNA amplification (PCR) assays. RT-PCR sizes are calculated from cDNA sequence data.

Primer Annealing Temperatures RT-PCR name Primer sequence (5' to 3') RT 1st RT 2 PCR size (bp) OnT25-F CAAAAATGCGTACCTTCATCGTTC 56°C 56°C 62°C 812 OnT25-R ATCATCAGCATCCGTAACGTG OnT23-F CGCAGCGCCCAGAATCGT 54°C 58°C 60°C 313 OnT23-R CAGCCTCACAACGGTGATGTC OnT3-F GCGATATGGCGAAATTCTTGG 50°C 50°C 62°C 800 OnT3-R T ATTAAACAGCAGTAGCCGCG OnCl-F CATTATGAAGTTCCTGGTCGTCCT 50°C 50°C 60°C 533 OnCl-Rl CGAAGGTCTCCTCCAGCTCA OnC2-F2 ACCGGCTCGAACAGCTTCG 56°C 56°C 62°C 463 OnC2-R GCATCAATCATTGGCTGCTCAA 163

Table 7.2. Ostrinia nubilalis midgut cDNA clones and putative derived mature protease properties.

Clone GenBank cDNA Length Molecular Putative name accession (bp) (aa) mass (kDa) Pi Protease type OnT25a AY953059 849 233 24.81 8.57 Trypsin OnT25b AY953060 864 233 24.81 7.78 Trypsin OnT25c AY953061 858 233 24.69 7.78 Trypsin OnT25d AY953062 862 233 24.74 8.58 Trypsin OnT25e AY953063 853 233 24.74 8.27 Trypsin T25 AY513649A 858 233 24.79 8.58 Trypsin OnT23a AY953068 821 209 22.67 5.95 Trypsin T23 AY513650a 975 222 23.68 6.22 Trypsin OnT3a AY953064 848 243 26.43 5.31 Trypsin OnT3b AY953065 849 243 26.41 5.78 Trypsin OnT3c AY953066 850 243 26.38 5.31 Trypsin OnT3d AY953067 850 241 25.89 4.96 Trypsin OnT3e AY953052 852 156s 16.94 6.40 Trypsin OnCl a AY953069 799 232^ NA NA Chymotrypsin OnClb AY953053 977 238 24.84 5.71 Chymotrypsin OnClc AY953054 983 238 24.71 5.48 Chymotrypsin OnCld AY953055 981 238 24.71 5.21 Chymotrypsin OnC2a AY953056 922 230 23.66 9.98 Chymotrypsin OnC2b AY953057 922 230 23.66 9.98 Chymotrypsin OnC2c AY953058 677 203^ NA NA Chymotrypsin A: Li et al. 2005 B: peptide truncation due to premature stop codon. C: partial sequence. NA: Data not available due to assumed partial sequence. Table 7.3. Pair-wise similarity matrix of O. nubilalis midgut expressed serine protease cDNA sequences. Three groups of trypsin-like (OnT25, OnT23, and OnT3) and two groups of chymotrypsin-like proteases (OnCl and OnC2) are indicated. Intraspecific cDNA sequence similarities are highlighted.

o ! 1 ! 1 1 ! O T25 OnT25a OnT25b OnT25c OnT3b OnClb OnClc OnCld o O o T23 O o OnT3d OnT3e OnC1a o OnC2b O T25 - 98 99 99 99 99 65 68 63 63 63 63 64 45 49 50 50 49 49 53 OnT25a 97 97 97 98 65 68 63 63 63 64 64 45 49 50 49 49 49 53 OnT25b - 99 99 99 65 68 63 63 63 64 64 45 49 49 49 49 49 53 OnT25c OnT25 group - 99 99 65 68 63 63 63 64 64 45 49 49 49 49 49 53

OnT25d - 99 65 68 63 63 63 64 64 45 49 49 49 49 49 53

OnT25e - 65 68 63 63 63 64 64 45 49 49 49 49 49 53

OnT23a - 83 73 73 73 71 71 46 45 44 44 34 34 41 T23 OnT23 group - 74 74 74 75 75 43 47 46 46 46 46 51

OnT3a - 98 88 97 96 36 40 40 40 37 37 36

OnT3b - 89 96 96 36 40 40 40 37 36 39

OnT3c - 90 91 32 36 36 36 32 32 40

OnT3d OnT3 group - 98 36 40 40 40 38 37 41

OnT3e - 36 40 40 40 38 38 40

OnC1a - 85 87 86 51 51 56

OnClb - 99 94 55 55 46

OnClc OnC1 group - 99 55 55 46

OnCld - 55 55 46 OnC2a - 98 76 OnC2b - 75 OnC2 group OnC2c - 165

Table 7.4. Test of Mendelian inheritance of trypsin and chymotrypsin alleles in O. nubilalis pedigrees PedlOb and Ped24a. PCR amplified genomic DNA fragments from

OnT23, OnT3, and OnCl genes were digested with HaeIII, and OnC2 with Cfol. Genotypic ratio among F2 offspring was compared to the expected Mendelian proportions based on parental genotypes. Significant allele frequency departures from Mendelian expectation were tested by chi-square (%2) analysis (significance at < 0.05). NP = no polymorphism.

Parental Observed F2 PCR-RFLP Expected Pedigree genotype genotypes F2 ratio I2 p-value (df) PedlOb OnT3 (3A11$A12 HI 1 (28): H12 (12): H22 (4) 9:6:1 2.22 0.3292 (2) OnCl c?Ell ÇE11 NP OnC2 (^C33$C34 C33 (8): C34 (34): C44 (0) 9:6:1 34.11 <0.0001 (2) Ped24a OnT23 c?H12$H23 HI 1(2): H12 (8): H22 (12): 1:4:4:4:2:1 7.00 0.2206 (5) H23 (12): H13 (1): H33 (5) OnT3 C^A13$A33 A33 (27): A13 (12): All (5) 9:6:1 3.27 0.1947 (2) OnCl SE22 ÇE11 Ell (10): E12 (23): E22 (15) 1:2:1 1.25 0.5698 (2) OnC2 c?H33$H33 NP Approximate sizes of observed PCR-RFLP fragments for alleles in pedigrees that showed Mendelian inheritance, and predicted but unobserved fragment sizes are within parentheses.

Gene Allele Fragments Gene Allele Fragments OnT3 Al: 470 and 460 bp OnCl El 740 A3: 340,300, 150, and 140 E2 490 and 240

OnT23 HI 1230 H2 780 and 490 H2 765,490, and (15) 166

CHAPTER 8. A (3-1,3-GALACTOSYLTRANSFERASE AND BRAINIAC/ BRE-5 HOMOLOG FROM OSTRINIA NUBILALIS

Brad S. Coates, Richard L. Hellmich, and Leslie C. Lewis

Abstract

Postradiational glycosylation of larval midgut epithelial peptide receptors is required prior to binding by Bacillus thuringiensis (Bt) Cry toxins. A 931 nt mRNA encoding a putative

297 residue |3-1,3-galactosyltransferase (P3GalT5) was isolated from O. nubilalis midgut tissue, and showed homology to Drosophila brainiac (bm) and Caenorhabditis elegans bre-5 peptides. DNA sequencing showed single nucleotide polymorphisms (SNPs) in the coding and promoter sequences of O. nubilalis (33GalT5 (On|33GalT5) alleles, and 3 of 31 CDS

SNPs were non-synonymous mutations. The mutant allele Onb3GalT5 I92 is detected by

Tsp5Q9l PCR-RFLP assay, was fixed within Cry1 Ac selected O. nubilalis, and present among < 0.78 % of wild individuals.

Keywords

Ostrinia nubilalis, single nucleotide polymorphism

1. Introduction

Bacillus thuringiensis crystalline (Cry) toxins bind glycosylphosphatidylinositol

(GPI)-anchored Lepidopteran midgut membrane receptors prior to pore formation (Lorance et al. 1997; Schnepf et al. 1998). The glycoprotein receptors of Cry toxins, aminopeptidase

N (APN; Knight et al. 1994), cadherin (Vadlamudi et al. 1993; Francis and Bulla 1995), and alkaline phosphatase receptors (de Maagd et al. 2001; Jurat-Fuentes et al. 2002) are N-acetyl- 167

D-galactosamine (GalNAc) modified, and may disproportionately be localized in cholesterol-

rich lipid rafts (Zhuang et al. 2002). GalNAc modification of anchored peptide receptors

may enhance toxin-receptor interactions (Knowles et al. 1991; Masson et al. 1995),

suggesting activated Cry toxins may associate with unrelated midgut receptor proteins that

share similar carbohydrate modifications. Furthermore, Cry1 Ac toxin binding may

additionally require association with glycolipids (Kumaraswami et al. 2001; Griffitts et al.

2005), especially ^-galactose termini of carbohydrate moieties.

Nematicidal toxins CrySB and Cryl4A share structural homology and invoke similar

physiological response as commercially used CrylA toxins, suggesting similar mode of

action (Griffitts et al. 2001). Caenorhabditis elegans is a model organism with extensive

genomic information available (The C. elegans Sequencing Consortium, 1998). Gut cells

from five recessive C. elegans Bt resistance {bre) mutants failed to take up CrySB and

Cryl4A toxins and evaded lysis, that likely was due to absence of membrane pores

(Maroquin et al. 2000; Griffitts et al. 2001). Complementation mapping identified a C.

elegans chromosome region containing a putative (3-1,3-galactosyltransferase family 5

member ((33GalT5; Amado et al 1999, Griffitts et al 2001, Hennet 2002) with homology to

Drosophila melanogaster BRAINIAC (Panin and Irvine 1998; Bruckner et al. 2000). Bre-5

and brn were shown to include iV-acetylglucosaminyltransferase activity in C. elegans

(Griffitts et al 2003) and D. melanogaster (Muller et al. 2002), respectively, that may add

terminal //-acetylgalactosamine (GalNAc) to oligosaccharides (Hennet 2002). The bre mutants demonstrated that lost or reduced function of posttranslational modification pathways could mediate resistance in nematodes. Structural similarities between insect and nematode-specific toxins, and requirements for carbohydrate binding prior to pore formation 168 in Lepidoptera suggest potential for a glycosylation pathway-mediated Bt resistance mechanism in insects (Griffitts et al. 2001; 2003).

Bacillus thuringiensis toxins may bind vV-acetyl-D-galactosamine modified ligands

(Knowles et al. 1991; Masson et al. 1995). The midgut membrane-bound glycoprotein cadherin was characterized from Lepidoptera (Francis and Bulla, 1997; Nagamatsu et al.,

1998 Gahan et al., 2001;Morin et al., 2003; Coates et al., 2005), and showed predicted N- glycosylation sites (Coates et al., 2005). Hypothesis that cadherin functioned as a candidate midgut Bt receptor came from transposon insertion-mediated cadherin knockout and reduction of CrylAa toxicity in H. virescens (Gahan et al., 2001), and linkage of Cry1 Ac resistance to cadherin allele segregation in P. gossypiella (Morin et al., 2003). Additionally, a glycosyl-phosphatidylinositol (GPI) anchored APN isoform with four potential N- glycosylation sites bound B. thuringiensis Cryl toxins in vitro (Knight et al. 1994). Role of

APN in Cryl Ac toxicity was demonstrated by transcript knockdown using RNA interference

(RNAi) in Spodoptera litura (Rajagopal et al., 2002), and removal of conserved N- glycosylation sites by block gene deletion in B. mori (Nakanishi et al. 1999). The Bt toxin domain III may interact with GalNAc moieties of APN (Jenkins et al. 1999, 2000), which is enhanced by association with cell membrane phosphatiylcholine (Sangadala et al. 2001). A third lepidopteran midgut receptor peptide, the GPI-anchored membrane-bound alkaline phosphatase, was identified from the YHD2 strain of resistance H. virescens (Jurat-Fuentes and Adang, 2004).

Unrelated midgut receptor proteins may share similar carbohydrate motifs bound by

Bt toxins. Altered expression of posttranslational modifiers that affect carbohydrate attachment may represent a general mode of resistance affecting multiple receptors (Griffitts 169 et al. 2001, 2003). Isolation and characterization of putative (3-1,3-galactosyltransferase genes from Lepidoptera are the first steps for determining roles, if any, in Bt resistance traits.

The European corn borer, Ostrinia nubilalis (Lepidoptera Crambidae), is an economically important model lepidopteran system. Glycoprotein receptors of O. nubilalis epithelial cells; a 220-kDa cadherin-like protein, and a 145-kDa or 154-kDa aminopeptidase (APN) isoforms bind Cryl Ab (Hua et al., 2001). Ostrinia nubilalis is a pest of maize, and has been controlled with foliar applications of the bacterium B. thuringiensis subsp. kurstaki.

Transgenic Cryl Ab toxin-expressing maize (Koziel et al., 1993) controls larval O. nubilalis

feeding (Walker et al., 2000). Field Resistance has not been detected, but moderate resistance has been found in O. nubilalis laboratory colonies (Bolin et al., 1999; Huang et al.,

1999). In 2003, 29% (22.9 million acres) of United States com acreage was commercial

Cryl Ab toxins expressing hybrids (USDA-NASS, 2003), suggesting a high selection

pressure and potential O. nubilalis adaptation.

2. Materials and Methods

2.1a Brainiac/BRE-5//33GalT5 Alignment and Phylogeny

A 345 residue consensus amino acid alignment was constructed for Drosophila

melanogaster gene CG4934-PA (GenBank NP 476901), Anopheles gambiae

ENSANGP00000007774 (GenBank XP 320943), and Caenorhabditis elegans BRE-5

(GenBank NM_171420) using Align X software (Informax, San Francisco, CA; gap penalty

= 10). Regions of peptide similarity were identified and corresponding cDNA sequence used to design a degenerate 3' RACE oligonucleotide b3GalT5-3pR (5'-TGG TAC GTT TCG

YTG GAG GAR TAY CC-3') with Primer3 (Rozen and Skaletsky, 1998). All phylogenetic 170 reconstruction methods used programs from the PHYLIP package (Felsenstein, 1989) with a

393 residue consensus amino acid alignment that also included homologous cDNA from

Homo sapiens, Mus musculus, Pongopygmaeus, and O, nubilalis (described below). One thousand bootstrap resampling steps were produced by the SeqBoot program, parsimony trees generated using ProtPars, a strict consensus tree estimated from all possible phytogenies with CONSENSE, and viewed using TreeView (Page, 1996).

2.1b Complementary DNA (cDNA) Synthesis and Cloning

(PT-AD; 5'-GGT GTA ATA CGA CGG CCT GGA ATT CTT TTT TTT TTT TTT TTT T-

3'), 2 |ig of total RNA, 40 U AMV reverse transcriptase (Promega, Madison, WI) in a 20 |_il reaction volume incubated at 42 °C for 30 m. Primer b3GalT5-3pR (5'-TGG TAC GTT

TCG YTG GAG GAR TAY CC-3') was designed using Primer3 (Rozen and Skaletsky,

1998) to anneal transcript sequence corresponding to conserved amino acids WYVSLEEYP.

Hot start PCR of first strand cDNA used 1 jal 1st strand cDNA product, 5 pmol each of primer b3GalT5-3pR and poly(T) adapter core primer (PT-Adcl; 5'-GTG TAA TAC GAC GGC

CTG G-3'), 1.0 U Tli proofreading polymerase (Promega), 100 fiM dNTPs, 1.5 mM MgCl%, and 2.5 (j.1 of 10X thermal polymerase buffer (Promega) in a 25 pi reaction volume. A PTC-

100 thermocycler (MJ Research, Watertown, MA) performed 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s for 40 cycles. Amplification was confirmed and fragment size estimated 171

by electrophoresis of 10 JLLI PCR product on a 10 cm 1.5 % agarose IX tris-borate EDTA gel

containing 0.5 jjg/ml ethidium bromide. PCR products were ligated using the pGEM-T easy

cloning system (Promega) by overnight incubation at 4 °C, and used to transform 80 (0.1 E.

coli SURE (Stratagene). DNA sequencing of plasmid inserts used dye-terminator cycle

sequencing (DTCS) quick-start kits (Beckman-Coulter, Fullerton, CA) and fragments

separated on a CEQ8000 capillary electrophoresis system (Beckman-Coulter). Insert

sequence was used to design a transcript-specific 5' RACE primer with Primer] (Rozen and

Skaletsky, 1998).

Oligonucleotide primer Onb3GalT5-Rl (5'-TGC TGC GGC ACT AAG CCC AC-3')

was designed from 3' RACE cDNA sequence and not to hybridize with First Choice RNA

Ligase-Mediated (RLM) RACE kit 5' adapter outer primer (5'-GCT GAT GGC GATG AAT

GAA CAC TG-3'; Ambion, Austin, TX). First Choice RLM RACE kit (Ambion) was used

to isolate 5' cDNA end of the O. nubilalis transcript according to manufacturer instructions;

RLM-RACE template was generated in 20 pi reactions with 1 pg of midgut total RNA.

Subsequent hot start PCR used 1.0 U Tli proofreading polymerase (Promega), 200 pM dNTPs, 1.5 mM MgClz, and 2.5 (il of 10 x thermal polymerase buffer (Promega), 1 pi RLM mRNA template, 5 pmol of primer Onb3GalT5-Rl, and 1 pi RLM-RACE 5' adapter outer primer (Ambion) in a 25 pi reaction. A PTC-100 thermocycler (MJ Research) cycled 40 times of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 m. RT-PCR products were, ligated, cloned, and sequenced as described previously. The reconstructed sequence of the brn and bre-5 homolog was called O. nubilalis |3-1,3-galactosyltransferase (Onb3Galt5). 172

2.2 Ostrinia nubilalis /33GalT5 Transcription

Larval O. nubilalis from the Corn Insects and Crop Genetics Research Unit laboratory colony were reared on artificial diet from neonate to 5th instar, in a constant environment (35 °C and 80% humidity). Total RNA was extracted from whole larvae, midgut, fat body, and head capsule with RNeasy extraction kits (Qiagen, Valencia, CA) and used a template for RT-PCR. Individual RT-PCR first strand cDNA reactions used 250 ng total RNA, 10 pmol of primer Onb3GalT5-Rl, 2.5 U Tth polymerase (Promega), 100 pM dNTPs, 2.5 mM MnClz, and 1.0 pi of 10X thermal polymerase buffer (Promega) in a 10 pi reaction volume. A PTC-100 thermocycler (MJ Research) performed a primer extension cycle of 85 °C for 1 m, 54 °C for 1 m, and 72 °C for 20 m. A 4.0 pi aliquot of 1st strand cDNA synthesis product was mixed with 1.6 pi chelate buffer (Promega), 10 pmol of primer b3GalT5-3pR (5'-TGG TAC GTT TCG YTG GAG GAR TAY CC-3'), MgCl2 to a concentration of 2.0 mM, and water to a final volume of 20 pi. RT-PCR reactions were carried out on a PTC-100 thermocycler (MJ Research) using 40 cycles of 95 °C for 30 s, 57

°C for 20 s, and 72 °C for 20s. RT-PCR products (20 pi) were separated on a 1 x 20 cm 6% polyacrylaminde (19:1 acrylaminde:bisacrylamide) IX tris-borate EDTA gel, stained by ethidium bromide, and digital images taken under UV illumination on a BioRad ChemiDoc

System (BioRad, Hercules, CA). Relative intensity of gel bands were measured using

Quantity One (v 4.5) 1-D Analysis Software (BioRad), with pixel intensity measured four independent times to get mean and standard error estimates.

2.3 The O. nubilalis (33GalT5 BAC and 5-UTR sequence 173

A pECBAC vector-based bacterial artificial chromosome (BAC) library was constructed from EcoBl digested O. nubilalis genomic DNA by Amplicon Express (Pullman,

WA) and transformed in E. coli strain XL1-Blue. A total of 18,432 clones were screened by

PCR with primers Onb3GalT5-F and Onb3GalT5-Rl using conditions described in section

2.4. The OnP3GalT5 locus was PCR amplified from a single BAC clone, OnB-2G17, and confirmed by DNA sequencing. The Onb3GalT5 promoter region and 5'-UTR were recovered using the TOPO Walker kit (Invitrogen, Carlsbad, CA) according to manufacturer instructions that included primer Onb3GalT5-Rl and 0.1 jag BAC clone OnB-2G17 template.

An approximately 1.5 kb fragment was amplified, purified using Qiaquick PCR spin columns

(Qiagen), and DNA sequenced as described previously individually using 1.6 pmol of primers Onb3GalT5-R2 (5'-CCT TCT TCG CCA GTA TGC C-3'), Onb3GalT5-5pSeq (5'-

ATG CGG CCT GGA ATC AAG TT-3') and Onb3GalT5-5pSeq (5'-TTG AAC GAC ATC

ATT GTC ACG-3').

Genomic DNA was isolated from 20 adult O. nubilalis (8 Cry9C selected) and 2 adult

Ostrinia furnicalis moths using DNAeasy isolation kit (Qiagen) according to manufacturer directions. A portion of the Onb3GalT5 promoter and 5'-UTR was PCR amplified using 5 pmol each of primers Onb3GalT5-3pUTR-F (5'-ACA TTG GTA GCA ACA AAA TTA

CAA-3') and Onb3GalT5-5pSeq. Reactions further contained 2.5 mM MgCla, 50 pM dNTPs, 0.9 U Taq DNA polymerase (Promega), and 100 ng of DNA template in a 12.5 pi reaction. PTC-100 thermocycler (MJ Research) conditions used 96 °C for 3 m, followed by

40 cycles of 96 °C for 30 s at, 60 °C for 30 s, and 72 °C for 40 s. PCR amplified products were purified using Qiaquick PCR spin columns (Qiagen), cloned into pGEM-T vector 174

(Promega), and DNA sequenced as described previously but using 1.6 pmol T7 primer. All sequences were aligned using Align X software (Informax) and point mutations identified.

2.4 Ostrinia nubilalis j.33GalT5 Single Nucleotide Polymorphism

Genomic DNA from the same 20 adult O. nubilalis and 2 adult Ostrinia furnicalis moths was used to amplify the Onb3GalT5 coding sequence (CDS). The Onb3GalT5 CDS was amplified using 2.5 mM MgCl?, 50 pM dNTPs, 7.5 pmol each of primers pmol

Onb3GalT5-Fl (5'-CGT GAC AAT GAT GTC GTT CAA-3') and Onb3GalT5-Rl, 0.9 U

Taq DNA polymerase (Promega), and 100 ng of DNA template in a 12.5 pi reaction. PTC-

100 thermocycler conditions used 95 °C for 2.5 m, followed by 40 cycles of 95 °C for 30 s,

62 °C for 30 s, and 72 °C for 1 m. PCR products purified using Qiaquick PCR spin columns

(Qiagen), and were sequenced as described previously, but sequenced in both directions using 1.6 pmol primer Onb3GalT5-Fl or Onb3GalT5-Rl. Sequence data were visually inspected for presence of hétérozygotes as overlapping fluorescent label peaks. Alignments were made among genomic and cDNA sequences using AlignX software (Informax; gap penalty = 5), and polymorphic nucleotide sites within restriction endonuclease sites identified. There were no introns in the genomic copy of O. nubilalis (3-1,3- galactosyltransferase Onb3GalT5.

Genotypic frequencies were determined from a Cryl Ac toxin selected colony, and

Mead, NE, South Shore, SD, Crawfordsville and Kanwaha, IA light trap samples. PCR products were digested with Mspl (Promega) and Tsp509l (New England BioLabs) for allele typing. PCR-RFLP reactions of fragments used PCR conditions described previously scaled 175 to 6.25 pl. Digestion reactions included 6.25 pi of PCR product, 2.5 pi lOx Buffer, 0.1 mg/pl BSA, and 0.25 U of Mspl and 0.125 U 7sp509I in 25 pi, and were incubated at 37 °C for 6 h then 65 °C for 14 h. Entire digest volumes were loaded onto 0.1 x 20 cm 8% polyacrylamide (19:1 acrylamide:bisacrylamide) gels, separated at 160 V for 6 h, and stained with ethidium bromide. Digital images were taken under UV illumination on a BioRad

ChemiDoc System (BioRad, Hercules, CA).

3. Results and Discussion

3.1 Ostrinia nubilalis f33GalT5 Gene Identification and Phylogeny.

The Drosophila neurogenic peptide encoding gene brainiac (brn) was identified as a (3-1,3-galactosyltransferase involved in posttranslational and lipid glycosylation (Amado et al., 1999; Schwientek et al., 2002; Muller et al. 2002). A brn ortholog in C. elegans bre-5 gene, showed a role in Cry5B and Cryl4A toxin resistance (Griffitts et al. 2001, 2003), and was placed in the (33GalT5 family of galactosyltransferases (Hennet 2002). The bre-5 transcripts from a mutant strain (ye17) have a predicted premature stop codon that truncates translation and eliminates a conserved catalytic motif (DDVFTGI; Hennet 2002), and is tightly linked to Bt resistance (Griffitts et al. 2001).

A 931 nt midgut-expressed mRNA was isolated from O. nubilalis with a single 297 amino acid open reading frame (ORF) that encoded a putative (3-1,3-galactosyl-transferase

(GenBank accession no. AY821557). The gene is referred to as O. nubilalis (3-1,3- galactosyl-transferase 5 (Onb3GalT5). A SilkBase search (http://www.ab.a.u- tokyo.ac.jp/silkbase/) indicated Onb3GalT5 homology to a, Bombyx mori prothoracic gland 176

expressed cDNA prgv0774 (E-value: 4 x 10"64), wing-disc-expressed cDNA weS00505 (E- value: 3 x 10~7), and epidermal-expressed cDNA epV30927 (E-value: 2 x lO"60).

Additionally, the inferred Onb3GalT5 peptide was similarity to D. melanogaster BRN

(GenBank: Q24157, BLASTp score = 149, E-value 10~35), C. elegans BRE-5 (GenBank:

NP_741492, BLASTp score = 99, E-value 6 x 10"20), and A. gambiae

ENSANGP00000007774 (GenBank: XP 320943, BLASTp score = 174, E-value 9 x 10"43).

A Protein Database (PDB) search using PredictProtein (Rost, 1996) demonstrated high similarity (S) and identity (IDE) of the Onb3GalT5 peptide to D. melanogaster BRAINIAC

(S = 54%, IDE = 34%, GenBank U41449), A. gambiae protein EBIG7774 (S = 59%, IDE =

39%; GenBank XM_320943). Additionally, proposed N-terminal transmembrane region

(TMR), N-glycosylation site, and semi-conserved Asp-Asp-Asp and Glu-Asp-Val-Tyr-Val-

Gly motifs present among |3-l,3-galactosyltransferases are present in the proposed O. nubilalis peptide.

Additionally seven highly conserved peptide domains among (3-1,3- galactosyltransferase family 5 members were identified in Onb3GalT5 (Fig. 1 single underline; Hennet 2002). A transmembrane region was predicted from amino acids 11 to 27 using the program Tmpred (http://www.ch.embnet.org/software/TMPRED _form.html; total score: 1840). This O. nubilalis transmembrane region (LLLCVCWISVYYLLGI) was similar to the homologous D. melanogaster brn region (LLLRCLLVLPLILLVDYCGL).

Although presumptive, cellular localization of Onb3GalT may be in the endoplasmic reticulum (30.4% probability; PSORT II prediction http://psort.nibb.ac.jp/form2.html) or golgi (26.6% probability). Most eukaryotic galactosyltransferases are anchored within the 177

Golgi (Chappel et al. 1994; Hennet 2002), but the hamster CGT (ceramide:UDP-galactose galaetosyltransferase) peptide is found in the endoplasmic reticulum of ovarian (CHO) cells

(Sprang et al. 1998). Empirical evidence is required to determine the cellular location of the

OnbSGalT peptide, however, since CGT was recognized as a distant member of the galaetosyltransferase family that we surmise Onb3GalT5 likely functions within the Golgi apparatus.

Identification of conserved motifs, and similarity to D. melanogaster BRN and C. elegans BRE suggests the O. nubilalis midgut-expressed cDNA (GenBank AY821557) encodes a family 5 (3-1,3-galactosyltransferase-like peptide (Hennet 2002). The gene was subsequently named Ostrinia nubilalis (3-1,3-galactosyltransferase 5 (Onb3GalT5). A 393 residue consensus amino acid alignment of seven (3-1,3-galactosyltransferase including O. nubilalis was used to construct a parsimony-based phylogeny rooted with the C. elegans

BRE-5 peptide (Griffitts et al. 2001; Fig. 2). The resulting tree suggested a strong division of the Onb3GalT peptide from other know vertebrate and invertebrate homologs. Phylogenetic separation may result from comparatively unique C-terminal truncation of the putative O. nubilalis (3-1,3-galactosyltransferase following the Glu-Asp-Val-Tyr-Val-Gly motif (Fig. 1).

3.2 Ostrinia nubilalis f33GalT5 Transcription

The Onb3GalT5 transcript was shown at high level in head and midgut-derived total

RNA samples (Fig. 3). Transcription was detected via presence of an approximately 180 bp

Onb3GalT5 RT-PCR product. Expression was investigated among four total RNA sources extracted from 5th instar O. nubilalis (midgut, fat body, head (brain), and whole larvae).

Total RNA level per reaction was standardized to 250 ng. Relative Onb3GalT5 transcript 178 levels appear highest in head-derived total RNA samples (mean 2070.7), followed by midgut samples (mean = 1463.3; Fig. 3). Transcript level in fat body may be lowest of all (mean =

1208.8), considering P-actin controls were nearly equally well amplified from each sample.

A decrease in band intensity observed from whole larvae RNA (mean = 1299.5) likely results from dilution of the target with low-expressing fat body-derived RNA.

Results suggest expression of the Onb3GalT5 transcript in head (possibly brain) and midgut tissues, and lower levels expressed in the fat body. The pattern of Onb3GalT5 transcriptional activation follows that observed from brn and bre-5. Total RNA was extracted from larval O. nubilalis heads, which contain many different tissues. Strong expression of brainiac, a P-1,3-galactosyltransferase, was observed in the brain of D. melanogaster. Therefore, O. nubilalis brain tissue from the head likely is the major contributor of Onb3GalT5 transcript, but high expression from mandibular muscle tissues cannot be ruled out. Transcription of Onb3GalT5 in midgut tissues was anticipated, due to midgut localization of BRE-5 in C. elegans (Griffitts et al. 2001). The C. elegans bre-5 enzymes are predicted to add carbohydrate modifications to cell membrane lipids and extracellular domains of proteins that may function as Bt toxin receptors in the midgut

(Griffitts et al. 2001, 2003).

3.3 The O. nubilalis (33 Ga IT5 5-UTR sequence

A portion of the O. nubilalis P-1,3-galaetosyltransferase (Onb3GalT5) promoter region sequence and the 5'-untranslated region was obtained from the BAC clone OnB-2G17

(GenBank Accession AY821558). A potential Onb3GalT5 transcriptional start site (+1 ;

TCAGGTC) was identified 37 to 44 nt upstream of the ATG start codon, which is similar to 179 the consensus initiator (inr) sequence (YYANWYY). Additionally, an Onb3GalT5 TATA box (A AT AAA) may be located at position -18 to -13 (Fig. 4). No other canonical promoter

elements were identified. A strong translational start site exists at position +43 (ATGR; ref).

An ATG is also located from +7 to +9, and is in-frame with the putative Onb3GalT5 ORF.

3.4 Ostrinia nubilalis f53GalT5 SNPs and population screening

A direct PCR product DNA sequencing strategy was used to identify Onb3Galt5 single nucleotide polymorphisms from O. nubilalis and O.furnicalis samples simultaneously

from both alleles. The strategy was feasible due to absence of introns from gDNA.

Heterozygous individuals were identified from overlapping signal of labeled dideoxy- nucleotides in electropherogram output (ref) from an 898 bp PCR product length. A total of

38 point mutations (SNPs) were observed from 13 O. nubilalis and 2 O.furnicalis individuals (30 alleles). Intraspecific comparison indicated 31 SNPs were present among a sample of 26 O. nubilalis alleles. Two different allele sequences were observed among

individuals from a Cryl Ac selected colony.

Three nonsynonymous changes were predicted from DNA sequence alignment (I92 to

V92, Q111 to K111, and D215 to N215). An I92 to V92 substitution exchanges two hydrophobic short chain aliphatic side chains that likely do not alter protein chemistry, and aligned homologous invertebrate (3-1,3-galactosyltransferases indicates this substitution is neutral

and takes place between species (Fig. 1). Replacements at positions 111 and 215 involve amino acids with polar hydrophilic side chains, although both exchanges induce different charges. Aspartic acid (Asp214) is a carboxylate anion at physiological pH, whereas the alternate amino acid has a uncharged asparagine residue (R214). Similarly the wild type 180 glutamine (Qm) is uncharged and the rare alternate amino acid, lysine (K11 '), is positively charged. The predicted pi of cytoplasmic domains for alternate amino acid forms I92KmN215

(6.42), [I/V]92QmN215 (6.19), and [W]92QmD215 (5.99) indicate all Onb3GalT5 allelic forms should remain partially soluble in the cytosol (pH approximately 7.5). Two polymorphic single nucleotide polymorphisms (SNPs) were detected by double restriction endonuclease digestion {Mspl and Tsp509l\ data not shown). Variation at these two sites may be used to genotypes in line crosses designed to follow segregation of particular genotypes with Bt resistance phenotypes.

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Walker KA, Hellmich RL, Lewis LC (2000) Late-instar European com borer (Lepidoptera: Crambidae) tunneling and survival in various transgenic com hybrids. J Econ Entomol 93:1276-1285.

Zhuang M, Oltean DI, Gomez I, Pullikuth AK, Soberon M, Bravo A, Gill SS (2002) Heliothis virescens and Manduca sexta lipid rafts are involved in Cryl A toxin binding to the midgut epithelium and subsequent pore formation. J Biol Chem 277:13863-13872. Figure 8.1. Peptide sequence alignment of invertebrate [3-1,3-galactosyltransferases (brainiac/BRE-5) homologs in Caenorhabditis elegans (Ce BRE-5; GenBank NM_171420), Drosophila melanogaster (Dm Brn; GenBank U41449), Anopheles gambiae (Ag XP 320943; gene ENSANGP00000007774; GenBank XP_320943), and Ostrinia nubilalis (Onb3GalT5). Transmembrane regions are double underlined, and highly conserved domains are underlined (Hennet 2002). Residues absolutely conserved are highlighted and conserved among insects are within dark boxes. Conserved Asp-X-Asp and variant Glu-Asp-Val- Tyr-Val-Gly motifs are underlined, and a conserved N-glycosylation site (Asn-Asn-Thr-[Leu or lie]) is overwritten with #. Also, - indicates a deletion within the alignment and * represents a stop codon. 1 80 Ce BRE-5 (1 MFLCVRILKRKYHELSSFQKLLIFTITIFLLWVLGWDKFRETSFG-DFSWPLETRNLOLRSKFTKYP Dm Brn (1 MQS KHRKLLLRCLLVLPLILLVDY CGLLTHLHELNFERH'FHYPLMDDTG SGSASS GLD KFAYLRVP S F Ag XP_3 2 0 943 (1 LAALVGLYLLQFFGAFTHFFEKDFESTFDYPLNGDILSDVYQLRHGQQPARPPINRYNYTYITDCEHK Onb3GalT5 (1 MMSFKRRKYKLLLCVCWISVYYLLGINDYWARRIEDGFDYPLNMDIOPLLOEVMAGKKPSVPPINYYPYRFLTNSG--

81 160 Ce BRE-5 (68 QCKFSGNGQKIIIIIIKSSAKNGPMRESVRKTWGVFRMIDGVEVMPIFIVG RVENMEIMRRIDVESEKYKDILAISD Dm Brn (69 TAEVPVDQPARLTMLIKSAVGNSRRREAIRRTWGYEGRFSDVHLRRVFLLG TAEDSEKDVAWESREHGDILQAEF Ag XP_32 0943 (69 CKEDDRLIAPRLVFWKSAIEHFDRRATIRKTWGYERRFSDVKIRTVFVLGRSRVHPNRRLQSLVDLESSTYRDIVQADE Onb3GalT5 (79 --KCNTVEKLDLFIVIKSAMDHFGHRNAVRLTYGKENLIPGRIVKSLFFVGIDESYPKSETQKKIDEEMVQFKDIIQIDF V Q 161 # 240 Ce BRE-5 (145 IDSYRNNTLKLFGAIDYAANPNQCSSPDFTFLVDDDYLVHIPNLVKFAKTK QKEELVYEGFVFDTSPFRLKIH Dm Brn (144 TDAYFNNTLK|MLGMRWAS--DQFNRSEFYLPVDDDYYVSAKNVLKFLGRG RQSHQPELLFAGHVFQTS PLRHKFS Ag XP_32 094 3 (149 VDDYFNNTIKTMTGFRWAV--SYCPRAKFYMFADDDFYVSAKNLLRYLMTE MELPPNVKLFSGFVFRSAPHRHRSS Onb3GalT5 (157 RDNYYNNTIKTMMSFRWVY--EHCNTADYYLPTDDDMYISVNNLLDYVHDKPVPTSTGHGNEQLYAGYVFE STPQRFITS D 241 320 Ce BRE-5 (218 KHSISLNEYPFSRYPPYVSAGAVFLTSETIA|FRNSIRKLKMFPFDDVFTGILAKTVMVAATHNENFIFWCRRVSQKEWD Dm Brn (218 KWYVSLEEYPFDRWPPYVTAGAFILSQKALRQLYAASVHLPLFRFDDVYLGIVALKAGISLQHCDDFRFHRPAYKGPDSY Ag XP_32 0 94 3 (223 KWYVSLEEYPWDMWPTYVTAGAFLLSHEALFEMYYVSMYTKHFRFDDIFLGIVAMKAGIEPLHSEEFYFHKAPYLGPQSY Onb3GalT5 (235 KWRVSLEEYPWSKWPSYVTAGAYWSNKSMKTMYAGSLFVKHFPFDDVFLGILAKKVGLVPAH*

321 350 Ce BRE-5 (295 DGVIAVHGYARKDLEYEYSQLNGFE* Dm Brn (2 95 S SVIASHE FGDPEEMTRVWNECRSANYA* Ag XP_32 094 3 (300 KYVLATHGYDEPTELTKVWNEIRASGYA* Onb3GalT5 (301 186

Figure 8.2. A consensus P-l,3-galactosyltransferase-5/brainiac/BRE-5 homolog phylogeny; constructed using parsimony from 1000 bootstrap pseudorepicates of a 393 residue peptide alignment.

CeNM171420

On Brn

AgXP320943 1000 443 DmU41449

322 MmBC063076

852 HsNM006057 1000 PpAB041417 187

Figure 8.3. Relative abundance O. nubilalis (3-1,3-galactosyltransferase (Onb3GalT5) transcripts among larval tissues, (mg = midgut; fb = fat body; hd = head; wb = whole body) from two total RNA samples (1 and 2). Fragment density plot of Onb3GalT5 indicates relative abundance of RT-PCR product using 250 ng of total RNA (mgl= 1543.3 ± 52.5; mg2 = 1383.3 ± 70.6; fbl -1135.2 ± 39.9; fb2 = 1282.4 ±31.2; hdl = 1889.6 ± 55.9; hd2 =

2251.73 ± 107.4; wbl 1375.1 ± 11.0; and wb2 = 1223.9 ± 1.1). The P-actin gene was used as a control for RNA integrity.

Density plot: Onb3GalT5 2500 .£ f'Vi -2000 |

1500 |

-1000 3. Onb3GalT5 jfcf mg1 mg2 fb1 fb2 hd1 hd2 wb1 wb2 Beta actin . * ,.s 188

Figure 8.4. Ostrinia nubilalis 0-1,3-galactosyltransferase (Onb3GalT5) 5-untranslated and promoter region sequence (BAC clone OnB-2G17; GenBank Accession AY821558).

Proposed Onb3GalT5 transcriptional start site is underlined (+1; TCAGGTC; consensus initiator (inr) sequence YYANWYY). A putative Onb3GalT5 TATA box (-18; AATAAA) is double underlined (consensus; TATAAA). Translational start (+43; ATGR) and N- terminal peptide sequence is highlighted. No other canonical promoter elements were identified.

-389 CAATTCAATGTTACATTGGTAGCAACAAAATTACAAACTACATGACTAACAATAGGTAAA >>>>>>>>>>>>>>>>>>>>>>>> -329 TAAATATCTGGAAAAGTGGTTTAGGATTTTTATAAGTTTATATAAAACAACACAATTATA

-269 ATGTGTTGTTTTCTTCTTTGTGTATTTATTTATGTATATTAAATTTTTAATTATTAGTAA

-209 ATAAATAGTGTTTAATTTAAGTATGTATTGTTAATTGTATAAACTTTTAAACCCTATATG

-14 9 ATGGATGAAGTAGTAATCCTGGTGCAAAGGCTCACTAATTGTTTAAATTGTTTTTACCTT

-089 TGGAAACTTATACTGGTTTTCTTGTTGCCTATCTTGTCATTATTATACACTGTAAAACCC

-029 TGGTTTTCCTAAATAAATAATATATTTTCAGGTCCATGTCCAGTAATGAAAAGAATCTCA + 1 +032 AGAACGTGACAATGATGTCGTTCAAAAGGAGAAAATACAAACTTTTGCTGTGTGTGTGTG MMSFKRRKYKLLLCVCV 189

CHAPTER 9. CONCLUSION

9.1 Introduction

Use of Bacillus thuringiensis (Bt) crystalline (Cry) toxins is an innovation that targeted insecticidal treatments, as opposed to broadcast chemical applications that indiscriminately eliminate all insects including beneficial predators and parisitoids.

Development of larval lepidopteran resistance to native or transgenic Bt Cryl A toxins is a threat to sustained effectiveness of targeted insecticide use. Reduced efficacy of Cryl Ab toxin against larval European corn borer, Ostrinia nubilalis, would reduce yield and producer income by billions of dollars.

The basis of resistance mechanisms has occurred at most points of the Bt toxin mode of action; toxin activation, degradation, or receptor binding. Molecular screening may detect geographic regions of increased insecticide resistance levels and are increasingly being incorporated into resistance diagnostics. Molecular assays that incorporate target genes have been developed to detect resistance genes in insect populations, including Cryl A resistant

Pectinophora. gossypiella cadherin alleles (Morin et al., 2003; Tabashnik et al., 2004).

Reduction in a trypsin-like protease mRNA levels was comparatively shown between resistant P. interpunctella (Oppert et al. 1997; Zhu et al. 2000) and O. nubilalis (Li et al.

2005) strains and isolines, but no genomic DNA assays were developed for mass screening.

The O. nubilalis cadherin was shown to be a receptor for Cryl Ab, Cryl Ac, and Cryl F (Hua et al. 2001; Flanagan et al. 2005). Preliminary data from O. nubilalis cadherin PCR-RFLP estimated high allelic diversity among individual susceptible adults collected near Mead, NE, and suggest assay use for molecular diagnostics (Coates et al. 2005a; Chapter 6). 190

9.2 Analysis of Bacillus thuringiensis resistance traits

One or more genes may mediate lepidopteran resistance to Cryl A toxins. Screening

Plutella xylostella for survival on Cryl A and Cryl F toxins suggested a single gene imparts

cross-resistance (Tabashnik et al. 1997), likely in a recessive manor (Meckel et al. 1999), and

additive gene action was predicted to mediate Bt resistance in Helicoverpa zea (Burd et al.

2003). Aminopeptidases initially were implicated as a major midgut receptor (Knight et al.

1994; Gill et al. 1995), followed by cadherin (Vadlamudi et al. 1995). Cadherin alleles were associated with resistance P. gossypiella phenotypes (Morin et al. 2003), and a transposon- mediated cadherin knockout was observed in a resistant Heliothis virescens strain (Gahan et al. 2001). Aminopeptidase transcript knockdown via RNA interference (RNAi) diminished larval Spodoptera litura susceptibility, reinforcing its role of in Bt resistance (Rajagopal et al.

2002). Inheritance of resistance in an O. nubilalis strain selected for survival on Dipel® Bt formulations was autosomal and incompletely dominant (Huang et al. 1999), and was shown to result from decreased levels of a trypsin-like protease mRNA, T23, in the midgut (Li et al.

2005). Data presented in Chapter 7 (Coates et al. 2005c In Press) describe Mendelian inheritance of a T23 Haelll genomic marker, which suggest its use for molecular diagnostics.

The T23 marker did show Mendelian inheritance pattern (Table 7.4). The marker will be applied in future studies involving inheritance of Bt toxin resistance phenotypes, and used in linkage mapping or studies to associate co-segregation of alleles with bioassayed O. nubilalis

Bt toxin resistance phenotypes.

Future investigation of Bt resistance mechanisms comprises several venues. First, the biological candidate approach uses prior organism or mechanistic knowledge to identify gene products that may affect resultant phenotypes. Simple comparison of between resistant and 191 susceptible strain coding sequences from candidate genes can show variation, but basis of the differences might be fixed interstrain differences due to random genetic drift encountered during selection as those that affect resistant phenotypes. Thus a more rational approach would attempt to associate segregation of fixed interstrain differences (alleles) with discrete

Bt resistance traits among F2 progeny derived from a resistant by susceptible parental crosses. Genotypes from multiple candidate gene loci would provide ability to partition portion of resistance traits imparted by different alleles when genotypes are compared between multiple pedigrees.

Secondly, a more broad genome-wide scanning approach would not look at particular genes, but follow segregation of anonymous genomic markers. High association of a particular allele with anonymous markers and Bt resistance phenotypes would be used to determine approximate genome position, and crossover frequency between adjacent markers are used to estimate distance (in centimorgan, cM) between the quantitative trait locus (QTL) and flanking markers. Assembly of overlapping bacterial artificial chromosome (BAC) into a physical (genomic) map can help identify single nucleotide polymorphisms (SNPs) in proximity to the QTL via end sequencing (accomplished by ordering BAC clones into a contiguous genomic section = contig). A saturated genetic map around QTL will narrow the genomic interval in which putative open reading frames (ORFs) must be identified and tested within the pedigrees.

Genomic approaches and expression studies can complement one another, whereas in isolation likely they provide only a partial view of biological events. Gene-by-gene quantitative real-time PCR comparing expression of biological candidate gene transcripts between resistant and susceptible lines are prone to detection of interstrain differences or 192 environmental differences (if field samples taken). Familywise analysis of single gene expression within an F2 pedigree structure can account for levels of genetic variation, but ignores epistatic (multigene) and upstream effects from various transcription factors.

Simultaneous multiple transcript expression analysis is possible using microarrays. These

"gene chips" are spotted in replicate with each transcript (cDNA or portion of transcript) from an organism or tissue. Hybridization of RNA samples from different treatment groups or genetic strains to represented transcripts may detect multiple fixed expression differences, some of which may have direct association with Bt resistance phenotypes. Secondary verification of differences between transcript levels should be undertaken by single gene quantitative real-time PCR to rule out differences resulting from interstrain, random treatment group, or environment effects.

9.3 Population genetics and the spread of Bacillus thuringiensis resistance traits

Resistance phenotypes may arise in multiple regions, but a more parsimonious scenario suggests resistance will occur in a single geographic location. Rates of migration between distinct subpopulations will influence movement of the local phenotype to other locations in North America. Prior knowledge of O. nubilalis movement was estimated from release and recapture trials (Showers et al. 2001) suggesting males move up to 32 km and females 25 km. Direct estimates of moth movement are dictated upon success of recapture, which becomes increasingly difficult with increasing distance (as the perimeter becomes insurmountably difficult to cover). Indirect estimates of migration rates may provide a more representative estimate of O. nubilalis movement in North America. 193

Ostrinia nubilalis molecular genetic markers from mitochondrial and genomic DNA

were presented in Chapter 3 and Chapters 4 and 5, respectively. These makers detect

variation in the North American population. Low but significant level of mitochondrial

haplotypes variation was observed between geographically distinct subpopulations (Fs-| <

0.024, P < 0.0001) and voltinism ecotypes (F$T < 0.03, P = 0.007), suggesting barriers to

gene flow exist (Coates et al. 2004; Chapter 3). Corroborating evidence was seen from

female haplotype data at two sex-linked microsatellite loci (Coates et al. 2003; Chapter 5), where a higher degree of geographic (FSj < 0.271, P < 0.0001) and voltinism ecotype

variation was estimated (F$T < 0.227, P < 0.0001). A large-scale population genetic study is

yet to be conducted using autosomal dinucleotide repeat microsatellite markers presented in

Chapter 4 (Coates et al. 2005b).

Migration rate (gene flow) among subpopulations and voltinism ecotypes will influence the frequency of resistant allele transfer and spread in North America, assuming the most parsimonious scenario whereby a highly resistant phenotype arises once. Migration rate estimates obtained from data presented in the aforementioned studies may influence computer models that predict the pattern and spread of resistance phenotypes in North

America. Temporal difference in adult emergence time suggests lower frequency of mating between compared to within ecotypes, and is supported by genetic variation detected using mitochondrial (Coates et al. 2003, Chapter 3; Coates et al. 2004, Chapter 3) and potentially using microsatellite markers (Coates et al. 2003; Chapter 5) in future studies.

Sympatic genetic differentiation of voltinism ecotypes may be based on temporally discontinuous mating periods. The data presented (Coates et al. 2004, Chapter 3; Coates et al. 2003; Chapter 5) used samples of pooled univoltine to pooled bivoltine moths. Attempts 194 were made to exclude individuals during any potential periods of uni- and bivoltine moth flight times. Future follow-up studies genotyping moths across an entire season could provide information regarding age structure and degree of univoltine/bivoltine mating period overlap. Genetic variation between sympatic O. nubilalis voltinism ecotypes could be further investigated using temporal autocorrelation of genotypes as a function of moth emergence time, and individual (ecotype) assignment based on likelihood analysis of variation genotypic distributions. In regions of voltinism ecotype overlap or sympatry three moth flight periods exist, a bivoltine first flight from late May thru mid June, a single univoltine period from late June to mid July, and finally a second flight of bivoltine moths from late July to mid August.

Tail ends of moth flight distributions overlap slightly between bi- and univoltine peaks depending on climatic conditions (Showers 1993), suggesting the potential for genetic exchange (inter-ecotype mating). F$T estimates derived from hierarchical tests of voltinism ecotype-based population subdivision (described earlier) provide an average level of genetic exchange between ecotypes, but the ultimate test of differentiation would be made in regions where ecotypes coexist. Temporal autocorrelation would be conducted using two binned groups, uni- and bivoltine moth samples, collected for the duration of the summer at four locations: Lamberton and Rosemount, MN, Center County, PA, and Dell Rapids, SD.

Genetic correlation between multilocus genotypes and corresponding voltinism designation will be made using Mantel tests, Moran's I (Weir 1999), and AMOVA (Smouse and Peakall

1999). Correlation will provide tests of the power of our genotyping system to differentiate voltinism ecotypes. 195

Since hybrids may be present, likelihood-based individual (ecotype) assignment tests

will be performed to identify potential "hybrid genomes" resulting from intercrossing. For

each genotyped individual, a Mendelian transmission probability gjkk> will be summed across

j loci containing alleles k and k' in subpopulation i; [T(g|i)= Oj T(gjkk>)]. Computation of the

natural log of transmission probabilities (ln(T(g|i)) will generate negative numbers. Initial

analysis will plot ln(T(g|univoltine) versus (ln(T(g|bivoltine) as a secondary means by which

to test our ability to identify sympatic individuals of different voltinism ectype assuming

respective genotypes will fall on opposite sides of a 45° line. Additionally, genotypes will be

plotted collectively for uni- and bivoltine ecotype designated genotypes, and should fall out

into distinct distributions. Simulated resampling of genotypes will be used to generate a

distribution of ln(T(g|univoltine) and ln(T(g|bivoltine) values, and individual genotypes from

our data set will be tested against a null hypothesis that they belong to their prescribed

ecotype designation, with a= 0.05 as the discriminatory point.

References

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ACKNOWLEDGEMENTS

I would like to thank the following

Program of Study Committee:

Daniel F. Voytas, Co-major Professor Leslie C. Lewis, Co-major Professor Richard L. Hellmich Fredric J. Janzen John D. Nason Jonathan F. Wendel

USDA-ARS Corn Insects & Crop Genetics Research Unit:

Douglas V. Sumerford Brendon and Kate Randy Ritland

Friends:

Drs. Jacko and "Dollar Dance" Dana Hartwigsen Kevin "Buddha" and Stephanie Lex Justin and Laura "the better half' Gibson Philbert and Rebecca Davis Ted and Julie Willis Trey Parker and Matt Stone, along with Stan, Kyle, Kenny and Carman The Griffins and the Quahog gang

Family:

Parents Doug and Eileen Coates: My constant cheerleaders who helped show me the right way to be.

Jacob Coates: Sorry you had to leave us so early.

Tyrell Firman: My little pal who puts fun into everyday life. I'll be with you every step on the road ahead.

Beth Harris: My kindred workaholic and perfectionist. Person with which I share hopes, dreams, and future.