The goal of this study was to investigate how plant selenium (Se) hyperaccumulation may affect ecological interactions and whether associated partners may affect Se hyperaccumulation. The Se hyperaccumulator Astragalus bisulcatus was collected in its natural seleniferous habitat, and x-ray fluorescence mapping and x-ray absorption near-edge structure spectroscopy were used to characterize Se distribution and speciation in all organs as well as in encountered microbial symbionts and herbivores. Se was present at high levels (704–4,661 mg kg−1 dry weight) in all organs, mainly as organic C-Se-C compounds (i.e. Se bonded to two carbon atoms, e.g. methylselenocysteine). In nodule, root, and stem, up to 34% of Se was found as elemental Se, which was potentially due to microbial activity. In addition to a nitrogen-fixing symbiont, the plants harbored an endophytic fungus that produced elemental Se. Furthermore, two Se-resistant herbivorous moths were discovered on A. bisulcatus, one of which was parasitized by a wasp. Adult moths, larvae, and wasps all accumulated predominantly C-Se-C compounds. In conclusion, hyperaccumulators live in association with a variety of Se-resistant ecological partners. Among these partners, microbial endosymbionts may affect Se speciation in hyperaccumulators. Hyperaccumulators have been shown earlier to negatively affect Se-sensitive ecological partners while apparently offering a niche for Se-resistant partners. Through their positive and negative effects on different ecological partners, hyperaccumulators may influence species composition and Se cycling in seleniferous ecosystems.

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.

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Until recently, the Old World bollworm (OWB) Helicoverpa armigera (Hübner) and the corn earworm Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) were geographically isolated. Both species are major pests of agricultural commodities that are known to develop insecticide resistance, and they now coexist in areas where H. armigera invaded the Americas. This is the first study to compare the susceptibility of the two species to conventional insecticides. The susceptibility of third instar H. armigera and H. zea larvae to indoxacarb, methomyl, spinetoram, and spinosad was determined using a diet-overlay bioassay in a quarantine laboratory in Puerto Rico. Mortality was assessed at 48 h after exposure for up to eight concentrations per insecticide. Spinetoram exhibited the highest acute toxicity against H. armigera, with a median lethal concentration (LC50) of 0.11 µg a.i./cm2, followed by indoxacarb and spinosad (0.17 µg a.i./cm2 for both) and methomyl (0.32 µg a.i./cm2). Spinetoram was also the most toxic to H. zea (LC50 of 0.08 µg a.i./cm2), followed by spinosad (0.17 µg a.i./cm2) and methomyl (0.18 µg a.i./cm2). Indoxacarb was the least toxic to H. zea, with an LC50 of 0.21 µg a.i./cm2. These findings could serve as a comparative reference for monitoring the susceptibility of H. armigera and H. zea to indoxacarb, methomyl, spinetoram, and spinosad in Puerto Rico, and may facilitate the detection of field-selected resistance for these two species and their potential hybrids in areas recently invaded by H. armigera.

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.

The Old World bollworm, Helicoverpa armigera (Hübner), is one of the most destructive agricultural pests worldwide. It was first recorded in Brazil in 2013, yet despite this recent introduction, H. armigera has spread throughout much of Latin America. Where H. armigera has become established, it is displacing or hybridizing with the congeneric New World pest Helicoverpa zea. In addition to the adaptive qualities that make H. armigera a megapest, such as broad range pesticide resistance, the spread of H. armigera in the New World may have been hastened by multiple introductions into South America and/or the Caribbean. The recent expansion of the range of H. armigera into the New World is analyzed herein using mtDNA of samples from South America, the Caribbean Basin, and the Florida Peninsula. Phylogeographic analyses reveal that several haplotypes are nearly ubiquitous throughout the New World and native range of H. armigera, but several haplotypes have limited geographic distribution from which a secondary introduction with Euro-African origins into the New World is inferred. In addition, host–haplotype correlations were analyzed to see whether haplotypes might be restricted to certain crops. No specialization was found; however, some haplotypes had a broader host range than others. These results suggest that the dispersal of H. armigera in the New World is occurring from both natural migration and human-mediated introductions. As such, both means of introduction should be monitored to prevent the spread of H. armigera into areas such as the United States, Mexico, and Canada, where it is not yet established.

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.

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