Marc E. Epstein

Integration of DNA barcoding into an ongoing inventory of complex tropical biodiversity

Inventory of the caterpillars, their food plants and parasitoids began in 1978 for todays Area de Conservacion Guanacaste (ACG), in northwestern Costa Rica. This complex mosaic of 120 000 ha of conserved and regenerating dry, cloud and rain forest over 0–2000 m elevation contains at least 10 000 species of non‐leaf‐mining caterpillars used by more than 5000 species of parasitoids. Several hundred thousand specimens of ACG‐reared adult Lepidoptera and parasitoids have been intensively and extensively studied morphologically by many taxonomists, including most of the co‐authors. DNA barcoding — the use of a standardized short mitochondrial DNA sequence to identify specimens and flush out undisclosed species — was added to the taxonomic identification process in 2003. Barcoding has been found to be extremely accurate during the identification of about 100 000 specimens of about 3500 morphologically defined species of adult moths, butterflies, tachinid flies, and parasitoid wasps. Less than 1% of the species have such similar barcodes that a molecularly based taxonomic identification is impossible. No specimen with a full barcode was misidentified when its barcode was compared with the barcode library. Also as expected from early trials, barcoding a series from all morphologically defined species, and correlating the morphological, ecological and barcode traits, has revealed many hundreds of overlooked presumptive species. Many but not all of these cryptic species can now be distinguished by subtle morphological and/or ecological traits previously ascribed to ‘variation’ or thought to be insignificant for species‐level recognition. Adding DNA barcoding to the inventory has substantially improved the quality and depth of the inventory, and greatly multiplied the number of situations requiring further taxonomic work for resolution.

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.

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.

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.

An Overview of the Lepidoptera (Insecta) of Plummers Island, Maryland

ABSTRACT Based on the examination of approximately 8100 specimens of Lepidoptera in the collection of the National Museum of Natural History and a review of relevant literature, we document 836 species in 488 genera and 48 families from Plummers Island, Maryland. Although the Lepidoptera are probably the best studied insect order on Plummers Island, data from the Washington, D.C. area indicate that there likely are many more microlepidoptera and butterflies on the site that are yet to be documented. Most families that were sampled adequately both historically (1901–1920) and in recent years (1998–2005) show a reduction in species richness and considerable species turnover. However, interpretation of these data is difficult owing to differences in sampling techniques and sampling frequency over the last 100 years.