A DNA Extraction Method for Insects From Sticky Traps: Targeting a Low Abundance Pest, Phthorimaea absoluta (Lepidoptera: Gelechiidae), in Mixed Species Communities.

Invasive insects can cause catastrophic damage to ecosystems and cost billions of dollars each year due to management expenses and lost revenue. Rapid detection is an important step to prevent invasive insects from spreading, but improvements in detection capabilities are needed for bulk collections like those from sticky traps. Here we present a bulk DNA extraction method designed for the detection of Phthorimaea absoluta Meyrick (Lepidoptera: Gelechiidae), an invasive moth that can decimate tomato crops. We test the extraction method for insect specimens on sticky traps, subjected to different temperature and humidity conditions, and among mock insect communities left in the field for up to 21 d. We find that the extraction method yielded high success (>92%) in recovering target DNA across field and lab trials, without a decline in recovery after three weeks, across all treatments. These results may have a large impact on tomato growing regions where P. absoluta is in the early stages of invasion or not yet present. The extraction method can also be used to improve detection capabilities for other bulk insect collections, especially those using sticky traps, to the benefit of pest surveys and biodiversity studies.

Genetic variation and population structure of Diaphorina citri using cytochrome oxidase I sequencing.

Citrus greening disease, or huanglongbing (HLB), is currently one of the most devastating diseases of citrus. The bacteria thought to be responsible for the disease, Candidatus Liberibacter asiaticus impact the majority of commercial citrus species worldwide. These bacteria are transmitted by the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, which is now found in most citrus growing regions. With no known cure, ACP-vectored HLB is responsible for significant economic losses to the global citrus industry. A better understanding of the global genetic diversity of D. citri would improve current and future pest management and mitigation programs. To assess the genetic diversity of D. citri in worldwide collections, a total of 1,108 sequences belonging to ACP gathered from 27 countries in the Americas, the Caribbean, Southeast and Southwest Asia were examined for the study. 883 D. citri came from 98 locations in 18 different countries, and were sequenced using a 678bp fragment of the mitochondrial cytochrome oxidase I (COI) gene. Additionally, 225 previously-reported D. citri COI sequences, were also included in our analysis. Analyses revealed 28 haplotypes and a low genetic diversity. This is in accordance with previous reports on the little diversity of D. citri in worldwide populations. Our analyses reveal population structure with 21 haplotypes showing geographic association, increasing the resolution for the source estimation of ACP. This study reveals the distribution of haplotypes observed in different geographic regions and likely geographic sources for D. citri introductions.

A Multiplex Real-Time PCR Assay for Screening Gypsy Moths (Lepidoptera: Erebidae) in the United States for Evidence of an Asian Genotype.

European gypsy moth populations (Lymantria dispar L.) are well established and a proven destructive force in hardwood trees throughout the United States and Canada. Introduction of the exotic Asian gypsy moth into North America would be even more impactful, as Asian gypsy moth populations have wider host ranges, and are capable of naturally dispersing more rapidly due to female flight ability. To support early detection and exclusion of Asian gypsy moth, the U.S. Department of Agriculture (USDA) uses molecular techniques to screen moths trapped in North America for evidence of common Asian genotype. In order to strengthen U.S. domestic capacity to screen moths quickly and efficiently, we report a real-time PCR assay for this pest. A probe system using TaqMan 5' nuclease chemistry is reported for detection of an allele associated with common Asian gypsy moth genotypes. The targeted allele is located at the nuclear FS1 locus currently used by the USDA in conventional PCR tests to screen for evidence of Asian gypsy moth introductions or introgression. The diagnostic probe is successfully multiplexed with a conserved 18S probe system to detect reaction failure due to poor sample quality or quantity. The specificity, sensitivity, and repeatability of the FS1-18S multiplex real-time PCR assay were tested on laboratory-reared and field-collected moths to demonstrate diagnostic utility. Implications of the new assay as a screening tool for evidence of Asian gypsy moth introgression and introduction are discussed.