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

Mammalian laryngseal air sacs add variability to the vocal tract impedance: Physical and computational modeling

Cavities branching off the main vocal tract are ubiquitous in nonhumans. Mammalian air sacs exist in human relatives, including all four great apes, but only a substantially reduced version exists in humans. The present paper focuses on acoustical functions of the air sacs. The hypotheses are investigated on whether the air sacs affect amplitude of utterances and∕or position of formants. A multilayer synthetic model of the vocal folds coupled with a vocal tract model was utilized. As an air sac model, four configurations were considered: open and closed uniform tube-like side branches, a rigid cavity, and an inflatable cavity. Results suggest that some air sac configurations can enhance the sound level. Furthermore, an air sac model introduces one or more additional resonance frequencies, shifting formants of the main vocal tract to some extent but not as strongly as previously suggested. In addition, dynamic range of vocalization can be extended by the air sacs. A new finding is also an increased variability of the vocal tract impedance, leading to strong nonlinear source-filter interaction effects. The experiments demonstrated that air-sac-like structures can destabilize the sound source. The results were validated by a transmission line computational model

Cq values plotted against end RFU values for each probe in the triplex real-time PCR assay.

<p>Cq values plotted against end RFU values for each probe in the triplex real-time PCR assay.</p

Alignment of ITS2 for <i>H</i>. <i>armigera</i> and <i>H</i>. <i>zea</i> showing sequence variation along with primer and probe locations.

<p>Alignment of ITS2 for <i>H</i>. <i>armigera</i> and <i>H</i>. <i>zea</i> showing sequence variation along with primer and probe locations.</p

Taxa sampled for study with country of origin, life stage, and number of specimens.

<p>Taxa sampled for study with country of origin, life stage, and number of specimens.</p