Todd M. Gilligan

Discovery of Lobesia botrana ([Denis & Schiffermüller]) in California: An Invasive Species New to North America (Lepidoptera: Tortricidae)

Abstract. The European grape vine moth, Lobesia botrana ([Denis and Schiffermüller]), is one of the most destructive pests of grape in the Palearctic Region. Larvae feed on fruit, causing direct damage and promoting secondary infection by Botrytis cinerea Persoon (botrytis bunch rot or gray mold). On September 30, 2009, tortricid larvae damaging grapes in the Napa Valley of California were identified as L. botrana, representing the first records of this species in North America. The presence of L. botrana could have a significant impact on California agriculture— wine, table, and raisin grapes are grown on more than 800,000 acres throughout the state. We provide descriptions and illustrations to aid in the identification of this newly arrived pest, along with a brief history of its discovery.

Patterns of mitochondrial haplotype diversity in the invasive pest Epiphyas postvittana (Lepidoptera: Tortricidae).

ABSTRACT The light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), is a horticultural pest of Australia and New Zealand that has more recently invaded Hawaii, Europe, and California. A 2,216-bp region of the mitochondrial genome containing the cytochrome oxidase I and II genes was sequenced from 752 individuals. Haplotype network analyses revealed a major split between a predominantly Western Australian clade and all other samples, suggestive of either a deep genetic divergence or a cryptic species. Nucleotide and haplotype diversity were highest in the country of origin, Australia, and in New Zealand populations, with evidence of haplotype sharing between New Zealand and Tasmania. Nucleotide and haplotype diversity were higher in California than within the British Isles or Hawaii. From the total of 96 haplotypes, seven were found in California, of which four were private. Within California, there have been at least two introductions; based on genetic diversity we were unable to assign a likely source for a single moth found and eradicated in Los Angeles in 2007; however, our data suggest it is unlikely that Hawaii and the British Isles are sources of the major E. postvittana population found throughout the rest of the state since 2006.

A Multiplex Real-Time PCR Assay to Diagnose and Separate Helicoverpa armigera and H. zea (Lepidoptera: Noctuidae) in the New World.

The Old World bollworm, Helicoverpa armigera (Hübner), and the corn earworm, H. zea (Boddie), are two of the most important agricultural pests in the world. Diagnosing these two species is difficult—adults can only be separated with a complex dissection, and larvae cannot be identified to species using morphology, necessitating the use of geographic origin for identification in most instances. With the discovery of H. armigera in the New World, identification of immature Helicoverpa based on origin is no longer possible because H. zea also occurs in all of the geographic regions where H. armigera has been discovered. DNA barcoding and restriction fragment length polymorphism (RFLP) analyses have been reported in publications to distinguish these species, but these methods both require post-PCR processing (i.e., DNA sequencing or restriction digestion) to complete. We report the first real-time PCR assay to distinguish these pests based on two hydrolysis probes that bind to a segment of the internal transcribed spacer region 2 (ITS2) amplified using a single primer pair. One probe targets H. armigera, the second probe targets H. zea, and a third probe that targets a conserved segment of 18S rDNA is used as a control of DNA quality. The assay can be completed in 50 minutes when using isolated DNA and is successfully tested on larvae intercepted at ports of entry and adults captured during domestic surveys. We demonstrate that the assay can be run in triplex with no negative effects on sensitivity, can be run using alternative real-time PCR reagents and instruments, and does not cross react with other New World Heliothinae.

Molecular Identification of the Light Brown Apple Moth (Lepidoptera: Tortricidae) in California Using a Polymerase Chain Reaction Assay of the Internal Transcribed Spacer 2 Locus

ABSTRACT A molecular protocol using a hemi-nested polymerase chain reaction (PCR) of the internal transcribed spacer region 2 (ITS2) is reported for the diagnosis of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in California. This protocol distinguishes the light brown apple moth from other moths in California based on size differences of PCR amplicons that are visualized on agarose gels. The molecular diagnostic tool generated no false negatives based on analysis of 337 light brown apple moths collected from California, Hawaii, England, New Zealand, and Australia. Analysis of a data set including 424 moths representing other tortricid species generated correct identification for >95% of the samples and only two false positives. Of the 761 moths tested only fourteen produced no PCR amplicons and five generated inconclusive data.

Discovery of false codling moth, Thaumatotibia leucotreta (Meyrick), in California (Lepidoptera: Tortricidae).

Abstract. The false codling moth, Thaumatotibia leucotreta (Meyrick), is one of the most destructive pests of avocado, citrus, and cotton in Africa. On July 24, 2008, a male of this species was identified from a pheromone trap located in Ventura County, California. Although larvae of T. leucotreta are frequently intercepted at U.S. ports-of-entry, primarily on bell peppers (Capsicum sp.), eggplant (Solanum melongena), and clémentines (Citrus sp.), this represents the first North American record outside of a port or international commercial shipment. Additional individuals have not been recorded from California suggesting that this species is not yet established in the state. We provide descriptions, illustrations, and other information to help in the identification of this species.

Combining Tpi and CO1 Genetic Markers to Discriminate Invasive Helicoverpa armigera From Local Helicoverpa zea (Lepidoptera: Noctuidae) Populations in the Southeastern United States

Abstract The recent establishment of the Old World pest Helicoverpa armigera (Hübner) into South America has had significant economic consequences and places the rest of the hemisphere at risk, emphasizing the need for improved methods of monitoring. A major complication is that a sibling species endemic to the New World, Helicoverpa zea (Boddie), is morphologically very similar, with the two species capable of producing fertile hybrids in the laboratory. The consequences of such hybridization in the field are uncertain, but could result in significant and unpredictable changes in the timing, range, and pesticide susceptibilities of Helicoverpa infestations. The objective here is to provide new genetic resources applicable to Helicoverpa populations in northern Florida and neighboring states (a region at risk for H. armigera) that can distinguish the two species and possible hybrids. The genetic variability in segments of the mitochondrial cytochrome oxidase 1 (CO1) and the Z-linked triosephosphate isomerase (Tpi) genes were determined for H. zea from the southeastern United States. These were compared to DNA sequences from H. armigera specimens from Morocco, Australia, and Europe. Phylogenetic network analysis showed a clear demarcation between the two species for all gene segments. These results extend earlier studies establishing CO1 as marker for discriminating the Helicoverpa species complex and introduce a new sex-linked genomic marker. The CO1 and Tpi markers in combination provide a more accurate and sensitive method than existing techniques for identifying hybridization between H. zea and H. armigera and could potentially be used to extrapolate the likely source of invasive H. armigera populations.

A Multiplex Real-Time Polymerase Chain Reaction Assay to Diagnose Epiphyas postvittana (Lepidoptera: Tortricidae)

ABSTRACT A molecular assay for diagnosis of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in North America is reported. The assay multiplexes two TaqMan real-time polymerase chain reaction (RT-PCR) probe systems that are designed to target DNA segments of the internal transcribed spacer region 2 (ITS2) and 18S rRNA gene. The RT-PCR probe designed for the 18S target recognizes a DNA sequence conserved in all of the moths included in the study and functions as a control in the assay. The second probe recognizes a segment of the ITS2 specifically found in E. postvittana and not found in the other moths included in the study, i.e., this segment is not conserved. Inclusion of the two markers in a single multiplex reaction did not affect assay performance. The assay was tested against 637 moths representing >90 taxa in 15 tribes in all three subfamilies in the Tortricidae. The assay generated no false negatives based on analysis of 355 E. postvittana collected from California, Hawaii, England, New Zealand, and Australia. Analysis of a data set including 282 moths representing 41 genera generated no false positives. Only three inconclusive results were generated from the 637 samples. Spike experiments demonstrated that DNA contamination in the assay can affect samples differently. Contaminated samples analyzed with the ITS2 RT-PCR assay and DNA barcode methodology by using the cytochrome oxidase I gene can generate contradictory diagnoses.

Diagnosis of Lobesia botrana (Lepidoptera: Tortricidae) Using Real-Time PCR.

A real-time PCR assay is reported for identification of Lobesia botrana (Denis and Schiffermüller) collected in California. This assay multiplexes two independent TaqMan probe systems in a single reaction tube to reduce handling time and sample exposure to environmental contaminants. One probe system targets a segment of DNA located in the internal transcribed spacer region 2 (ITS2) that is present in the L. botrana genome but absent in native North American Tortricidae. The second probe system serves as a control for DNA quality by targeting a segment of the 18S rDNA gene that is conserved in L. botrana and all of the tested nontarget species. The assay successfully diagnosed 70 Lobesia botrana specimens and 95 nontarget specimens. No false-positive or false-negative results were observed supporting its application for identification of this pest in California.

A droplet digital PCR (ddPCR) assay to detect Helicoverpa armigera (Lepidoptera: Noctuidae) in bulk trap samples

Moths in the genus Helicoverpa are some of the most important agricultural pests in the world. Two species, H. armigera (Hübner) and H. zea (Boddie), cause the majority of damage to crops and millions of dollars are spent annually on control of these pests. The recent introduction of H. armigera into the New World has prompted extensive survey efforts for this species in the United States. Surveys are conducted using bucket traps baited with H. armigera pheromone, and, because the same pheromone compounds attract both species, these traps often capture large numbers of the native H. zea. Adult H. armigera and H. zea are very similar and can only be separated morphologically by minor differences in the genitalia. Thus, a time consuming genitalic dissection by a trained specialist is necessary to reliably identify either species, and every specimen must be dissected. Several molecular methods are available for differentiating and identifying H. armigera and H. zea, including two recently developed rapid protocols using real-time PCR. However, none of the published methods are capable of screening specimens in large batches. Here we detail a droplet digital PCR (ddPCR) assay that is capable of detecting a single H. armigera in a background of up to 999 H. zea. The assay has been tested using bulk extractions of 1,000 legs from actual trap samples and is effective even when using poor quality samples. This study provides an efficient, rapid, reproducible, and scalable method for processing H. armigera survey trap samples in the U.S. and demonstrates the potential for applying ddPCR technology to screen and diagnose invasive species.

A Real-Time PCR Assay for the Separation of Autographa gamma (Noctuidae: Plusiinae) From Morphologically Similar Species in North America

Abstract The silverY moth, Autographa gamma L. (Noctuidae: Plusiinae), is a pest of major economic importance in its native range of Europe, Asia, and North Africa. Although not present in North America, larvae of A. gamma are commonly intercepted in commodity shipments at U.S. ports, and adult surveys are conducted each year in more than 20 states. Because of the similarity of A. gamma to several native North American species that are attracted to the same pheromone lure, morphological identification of adults is difficult and requires dissection. In 2010, a specimen of Autographa californica (Speyer, 1875) (Lepidoptera: Noctuidae) from Pennsylvania was incorrectly identified as A. gamma, signaling the need for an alternative method of rapid identification. Here we detail a real-time PCR assay capable of identifying A. gamma specimens in approximately 45 min using extracted DNA. The assay uses a hydrolysis probe that targets a species-specific segment of the CO1 DNA barcode region, while a control probe targets a conserved region of 18S rDNA. The assay was tested with two independent runs of 452 specimens of Plusiinae representing 23 different species. The assay provided unambiguous data 99.7% of the time and did not result in any false positives; these data were used to develop a rule set for interpreting the real-time PCR results. In addition, the same diagnostic probe was tested in bulk sample simulations using real-time PCR and droplet digital PCR where A. gamma could be detected in concentrations as low as 1:1,000,000 (gamma:californica).These experiments provide baseline data for developing a bulk sample assay.