Detection of Khapra Beetle Environmental DNA Using Portable Technologies in Australian Biosecurity.

Khapra beetle, Trogoderma granarium Everts, 1898, is a serious pest of stored grain products globally. Environmental DNA (eDNA)-based methods offer sensitive detection tools used to inform biosecurity officers on the presence of high-risk pests. This study tested laboratory and portable molecular technologies to detect khapra beetle environmental DNA extracted from dust samples collected during biosecurity responses (Tuggeranong and Fyshwick) to khapra beetle incursions in Australia. Airborne and floor dust samples were collected opportunistically using handheld vacuum cleaners and eDNA was extracted using either field or laboratory-based extraction methods and analyzed using laboratory benchtop real time PCR machines and portable machines with two TaqMan and one LAMP-based assay. We successfully collected, extracted, and amplified khapra beetle eDNA from dust samples by qPCR, but failed to amplify T. granarium eDNA using LAMP. The Laboratory qPCR machine showed significantly higher mean Ct values (p < 0.001) and significantly higher positive detections for both assays (p < 0.001) compared to the portable thermocycler. DNA yield was significantly higher in samples extracted using laboratory-based kits compared to field kits (p < 0.001) for both vacuumed and airborne samples (Mean DNA ± S.D. = 5.52 ± 4.45 and 4.77 ± 1.68 ng/µL, respectively), compared to field kits, (1.75 ± 1.17 and 1.36± 1.29 ng/µL for vacuumed and airborne samples, respectively). There were no significant differences in DNA yield between collection methods or differences in amplification associated to extraction or collection methods in either platform tested in this study. Portable technologies tested in this study (Franklin™ Real Time Thermocycler and Genie III) accurately amplified all tissue derived DNA during assay optimisation and field testing, highlighting the capacity of these technologies to complement biosecurity in confirming specimen ID. There was a high incidence of positive detections in field negative controls (Tuggeranong = 12.3 % and Fyshwick = 50 %), mostly attributed to the use of contaminated vacuum cleaners. We discuss suitable methods to minimize sample cross-contamination, the potential of portable molecular technologies as tools for biosecurity applications, and the suitability of eDNA-based molecular detection methods to complement global trade biosecurity for one of the most invasive and important grain pests worldwide.

A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes.

The rapid and accurate identification of invertebrate pests detected at the border is a challenging task. Current diagnostic methods used at the borders are mainly based on time consuming visual and microscopic examinations. Here, we demonstrate a rapid in-house workflow for DNA extraction, PCR amplification of the barcode region of the mitochondrial cytochrome oxidase subunit I (COI) gene and Oxford Nanopore Technologies (ONT) MinION sequencing of amplified products multiplexed after barcoding on ONT Flongle flow cells. A side-by-side comparison was conducted of DNA barcode sequencing-based identification and morphological identification of both large (>0.5 mm in length) and small (<0.5 mm in length) invertebrate specimens intercepted at the Australian border. DNA barcode sequencing results supported the morphological identification in most cases and enabled immature stages of invertebrates and their eggs to be identified more confidently. Results also showed that sequencing the COI barcode region using the ONT rapid sequencing principle is a cost-effective and field-adaptable approach for the rapid and accurate identification of invertebrate pests. Overall, the results suggest that MinION sequencing of DNA barcodes offers a complementary tool to the existing morphological diagnostic approaches and provides rapid, accurate, reliable and defendable evidence for identifying invertebrate pests at the border.

Relationship between persistent organic pollutants (POPs) and ranging patterns in common bottlenose dolphins (Tursiops truncatus) from coastal Georgia, USA.

Bottlenose dolphins (Tursiops truncatus) are apex predators in coastal southeastern U.S. waters; as such they are indicators of persistent organic pollutants (POPs) in coastal ecosystems. POP concentrations measured in a dolphin's blubber are influenced by a number of factors, including the animal's sex and ranging pattern in relation to POP point sources. This study examined POP concentrations measured in bottlenose dolphin blubber samples (n=102) from the Georgia, USA coast in relation to individual ranging patterns and specifically, distance of sightings from a polychlorinated biphenyl (PCB) point source near Brunswick, Georgia. Dolphin ranging patterns were determined based upon 5years of photo-identification data from two field sites approximately 40km apart: (1) the Brunswick field site, which included the Turtle/Brunswick River Estuary (TBRE), and (2) the Sapelo field site, which included the Sapelo Island National Estuarine Research Reserve (SINERR). Dolphins were categorized into one of three ranging patterns from photo-identification data. Individuals with sighting histories exclusively within one of the defined field sites were considered to have either Brunswick or Sapelo ranging patterns. Individuals sighted in both field sites were classified as having a Mixed ranging pattern. Brunswick males had the highest concentrations of PCBs reported for any marine mammal. The pattern of PCB congeners was consistent with Aroclor 1268, a highly chlorinated PCB mixture associated with a Superfund site in Brunswick. PCB levels in Sapelo males were lower than in Brunswick males, but comparable to the highest levels measured in other dolphin populations along the southeastern U.S. Female dolphins had higher Aroclor 1268 proportions than males, suggesting that the highly chlorinated congeners associated with Aroclor 1268 may not be offloaded through parturition and lactation, as easily as less halogenated POPs. Individuals sighted farther from the Superfund point source had lower Aroclor 1268 proportions.