Wee Tek Tay

A Brave New World for an Old World Pest: Helicoverpa armigera (Lepidoptera: Noctuidae) in Brazil

The highly polyphagous Old World cotton bollworm Helicoverpa armigera is a quarantine agricultural pest for the American continents. Historically H. armigera is thought to have colonised the American continents around 1.5 to 2 million years ago, leading to the current H. zea populations on the American continents. The relatively recent species divergence history is evident in mating compatibility between H. zea and H. armigera under laboratory conditions. Despite periodic interceptions of H. armigera into North America, this pest species is not believed to have successfully established significant populations on either continent. In this study, we provide molecular evidence via mitochondrial DNA (mtDNA) cytochrome oxidase I (COI) and cytochrome b (Cyt b) partial gene sequences for the successful recent incursion of H. armigera into the New World, with individuals being detected at two sites (Primavera do Leste, Pedra Preta) within the State of Mato Grosso in Brazil. The mtDNA COI and Cyt b haplotypes detected in the Brazilian H. armigera individuals are common throughout the Old World, thus precluding identification of the founder populations. Combining the two partial mtDNA gene sequences showed that at least two matrilines are present in Brazil, while the inclusion of three nuclear DNA Exon-Primed Intron-Crossing (EPIC) markers identified a further two possible matrilines in our samples. The economic, biosecurity, resistance management, ecological and evolutionary implications of this incursion are discussed in relation to the current agricultural practices in the Americas.

Mitochondrial DNA analysis of field populations of Helicoverpa armigera (Lepidoptera: Noctuidae) and of its relationship to H. zea

BackgroundHelicoverpa armigera and H. zea are amongst the most significant polyphagous pest lepidopteran species in the Old and New Worlds respectively. Separation of H. armigera and H. zea is difficult and is usually only achieved through morphological differences in the genitalia. They are capable of interbreeding to produce fertile offspring. The single species status of H. armigera has been doubted, due to its wide distribution and plant host range across the Old World. This study explores the global genetic diversity of H. armigera and its evolutionary relationship to H zea.ResultsWe obtained partial (511 bp) mitochondrial DNA (mtDNA) Cytochrome Oxidase-I (COI) sequences for 249 individuals of H. armigera sampled from Australia, Burkina Faso, Uganda, China, India and Pakistan which were associated with various host plants. Single nucleotide polymorphisms (SNPs) within the partial COI gene differentiated H. armigera populations into 33 mtDNA haplotypes. Shared haplotypes between continents, low F-statistic values and low nucleotide diversity between countries (0.0017 – 0.0038) suggests high mobility in this pest. Phylogenetic analysis of four major Helicoverpa pest species indicates that H. punctigera is basal to H. assulta, which is in turn basal to H. armigera and H. zea. Samples from North and South America suggest that H. zea is also a single species across its distribution. Our data reveal short genetic distances between H. armigera and H. zea which seem to have been established via a founder event from H. armigera stock at around 1.5 million years ago.ConclusionOur mitochondrial DNA sequence data supports the single species status of H. armigera across Africa, Asia and Australia. The evidence for inter-continental gene flow observed in this study is consistent with published evidence of the capacity of this species to migrate over long distances. The finding of high genetic similarity between Old World H. armigera and New World H. zea emphasises the need to consider work on both pests when building pest management strategies for either.

Complete rRNA Gene Sequences Reveal that the Microsporidium Nosema bombi Infects Diverse Bumblebee (Bombus spp.) Hosts and Contains Multiple Polymorphic Sites

Abstract. Characterisation of microsporidian species and differentiation among genetic variants of the same species has typically relied on ribosomal RNA (rRNA) gene sequences. We characterised the entire rRNA gene of a microsporidium from 11 isolates representing eight different European bumblebee (Bombus) species. We demonstrate that the microsporidium Nosema bombi infected all hosts that originated from a wide geographic area. A total of 16 variable sites (all single nucleotid polymorphisms (SNPs)) was detected in the small subunit (SSU) rRNA gene and 42 (39 SNPs and 3 indels) in the large subunit (LSU) rRNA sequence. Direct sequencing of PCR‐amplified DNA products of the internal transcribed spacer (ITS) region revealed identical sequences in all isolates. In contrast, ITS fragment length determined by PAGE and sequencing of cloned amplicons gave better resolution of sequences and revealed multiple SNPs across isolates and two fragment sizes in each isolate (six short and seven long amplicon variants). Genetic variants were not unique to individual host species. Moreover, two or more sequence variants were obtained from individual bumblebee hosts, suggesting the existence of multiple, variable copies of rRNA in the same microsporidium, and contrary to that expected for a class of multi‐gene family under concerted evolution theory. Our data on within‐genome rRNA variability call into question the usefulness of rRNA sequences to characterise intraspecific genetic variants in the Microsporidia and other groups of unicellular organisms.

Multiple rRNA variants in a single spore of the microsporidian Nosema bombi

ABSTRACT. To understand the source of the multiple DNA sequence variants of Nosema bombi ribosomal RNA (rRNA) found in a single bumble bee host, we PCR amplified, cloned, and sequenced the partial rRNA gene from 125 clones, which were derived from four out of 46 spores individually isolated from a single host by laser microdissection. At least two rRNA variants, characterized by either (GTTT)2 or (GTTT)3 repeat units within the internal transcribed spacer (ITS) region, were found per spore in approximately equal proportions, variants which were also found in approximately equal proportions in 55 clones of the two DNA extracts of multiple spores from the same host. Firstly, we demonstrate for the first time that DNA sequences can be obtained from single‐binucleate microsporidia. Secondly, it appears that concerted evolution has not homogenized the sequences of all rRNA copies within a single N. bombi spore or even within a single nucleus. We thereby demonstrate unequivocally that two or more rRNA sequence variants exist per N. bombi spore, and urge caution in the use of multicopy rRNA genes for population genetic and phylogenetic analysis of this and other Microsporidia unless homologous copies can be reliably typed.

The potential distribution of invading Helicoverpa armigera in North America: Is it just a matter of time?

Helicoverpa armigera has recently invaded South and Central America, and appears to be spreading rapidly. We update a previously developed potential distribution model to highlight the global invasion threat, with emphasis on the risks to the United States. The continued range expansion of H. armigera in Central America is likely to change the invasion threat it poses to North America qualitatively, making natural dispersal from either the Caribbean islands or Mexico feasible. To characterise the threat posed by H. armigera, we collated the value of the major host crops in the United States growing within its modelled potential range, including that area where it could expand its range during favourable seasons. We found that the annual value of crops that would be exposed to H. armigera totalled approximately US

Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein

78 billion p.a., with US

Morphological and molecular characters reveal differentiation in a Neotropical social bee, Melipona beecheii (Apidae: Meliponini)

843 million p.a. worth growing in climates that are optimal for the pest. Elsewhere, H. armigera has developed broad-spectrum pesticide resistance; meaning that if it invades the United States, protecting these crops from significant production impacts could be challenging. It may be cost-effective to undertake pre-emptive biosecurity activities such as slowing the spread of H. armigera throughout the Americas, improving the system for detecting H. armigera, and methods for rapid identification, especially distinguishing between H. armigera, H. zea and potential H. armigera x H. zea hybrids. Developing biological control programs, especially using inundative techniques with entomopathogens and parasitoids could slow the spread of H. armigera, and reduce selective pressure for pesticide resistance. The rapid spread of H. armigera through South America into Central America suggests that its spread into North America is a matter of time. The likely natural dispersal routes preclude aggressive incursion responses, emphasizing the value of preparatory communication with agricultural producers in areas suitable for invasion by H. armigera.

Molecular markers to discriminate among four pest species of Helicoverpa (Lepidoptera: Noctuidae)

The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode-of-action of the two Bt Cry toxins.

Population Genetic Structure of the Cotton Bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in India as Inferred from EPIC-PCR DNA Markers

Morphometrics and DNA microsatellites were used to analyse the genetic structure of populations of the stingless bee M. beecheii from two extremes of its geographic range. The results showed that populations from Costa Rica and Yucatan exhibit substantial phenotypic and molecular differentiation. Bees from Yucatan were smaller and paler than those from Costa Rica. The value of multilocus FST = 0.280 (P < 0.001) confirmed that there were significant molecular genetic differences between the two populations. Populations showed significant deviation from Hardy Weinberg equilibrium and the values of FIS (the inbreeding coefficient) were positive for Costa Rica = 0.416 and the Yucatan Peninsula = 0.193, indicating a lack of hétérozygotes in both populations possibly due to inbreeding. The DNA sequence of 678 bp of the mitochondrial gene COI differed between populations by 1.2%. The results of this study should be considered in conservation programmes, particularly with regard to the movement of colonies between regions.ZusammenfassungDie über 400 Arten umfassenden pantropisch verbreiteten Meliponini sind vergleichsweise wenig untersucht, insbesondere gibt es nur wenige Studien zur Populationsstruktur. Die Art Melipona beechei weist eine geographische Verbreitung von Mexiko bis Costa Rica auf (Abb. 1), so dass es möglich ist, dass lokal adaptierte Ökotypen existieren. Wir nutzen einen morphometrischen Ansatz, sowie DNA Mikrosatellitenloci und Gensequenzen mitochondrialer DNA um die Populationsstruktur von M. beecheii an den Extrempunkten der geographischen Verbreitung zu untersuchen.Die Bienen wurden 1998 an sieben Orten auf der Halbinsel Yucatan (65 Völker) und an einem Ort in Costa Rica (15 Völker) gesammelt. Elf Morphometriemerkmale am Kopf, an Flügeln und Beinen wurden an 10–12 Arbeiterinnen pro Volk vermessen und die einzelnen Standorte wurden mittels ANOVA (gefolgt von Tukey post hoc Tests) verglichen. Populationsunterschiede in Farbmarken am Kopf (Abb. 2) wurden mittels Kontingenz G-Test herausgearbeitet. Anschliessend wurde eine Hauptkomponentenanalyse (PCA auf der Basis einer Korrelationsmatrix) mit allen log-transformierten metrischen Merkmalen durchgeführt.Sechs Mikrosatellitenloci (T4, T7, Mbill, Mb201, B116 und B124) wurden an jeweils einer Arbeiterin pro Volk untersucht. Zusätzlich wurden 678 Basenpaare des mitochondrialen Gens COI für jeweils zwei Individuen aus Costa Rica und Yucatan sequenziert. Die Verknüpfung (linkage) der Mikrosatellitenloci wurde mittels des Programmpakets GENEPOP Version 3.1. getestet. Die Allelfrequenzen und beobachtete sowie Nei’s (1978) unabhängige Erwartungswerte für Heterozygotie an jedem Mikrosatellitenlocus wurden für jede Population mittels des FSTAT Programmpakets ermittelt. Die effektive Anzahl an Allelen (na) wurde als na = 1/gSpi2 berechnet, wobei pi die Frequenz des i-ten Allels darstellt. Abweichungen vom Hardy-Weinberg-Gleichgewicht an jedem Locus und für jede Population wurden mittels in GENEPOP verfügbaren exakten Tests in einem Markov Ketten-Ansatz ermittelt. Der unabhängige Multilocus-Schätzwert für FST zwischen Populationen wurde benutzt, um die Signifikanz der genetischen Differenzierung mittels FSTAT herauszuarbeiten und um den jeweiligen Inzuchtgrad FIS und seine Konfidenzintervalle zu berechnen.Die Populationen aus Costa Rica und Yucatan zeigten eine erhebliche phänotypische und genetische Differenzierung. Dabei bildeten die Völker aus Yucatan eine homogene Gruppe, die sich deutlich (als kleiner und weniger farbkräftig) von den Costa Rica Völkern unterschied (Tab. I, II; Abb. 3). Der Multilocus Wert für FST = 0,280 (P < 0,001) belegt die signifikante molekulargenetische Differenzierung zwischen den beiden Populationen (Tab. IV). Beide Populationen zeigten erhebliche Abweichungen vom Hardy-Weinberg Gleichgewicht. Die FIS — Werte für Costa Rica = 0,416 (P < 0,05) und Yucatan = 0,193 (P < 0,05) waren hochsignifikant, was auf ein vermutlich inzuchtbedingtes Fehlen an Heterozygoten hinweist. In den DNA-Sequenzen für COI unterschieden sich die beiden Populationen um 1,2 %. Die Ergebnisse der vorliegenden Studie sollten in Konservierungsprogramme Eingang finden, vor allem dann, wenn der Austausch von Völkern zwischen Regionen in Betracht gezogen wird.

Bt resistance in Australian insect pest species.

The four significant pest species in the Helicoverpa genus (H. armigera, H. assulta, H. punctigera and H. zea) are morphologically similar and can only be reliably distinguished through dissection of adult genitalia. Two partial regions of the mitochondrial DNA (mtDNA), the cytochrome oxidase subunit I (COI) and the cytochrome b (Cyt b) genes were amplified by PCR and digested with restriction endonucleases. The restriction patterns, generated by the endonucleases BstZ17I and HphI, demonstrated reliable differentiation of the four Helicoverpa pest species. This technique is fast, reliable and effective at distinguishing specimens irrespective of their life stages and offers support to conventional taxonomic differentiation based on morphological characters.