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.

No link between nymph and adult coloration in shield bugs: weak selection by predators.

Many organisms use different antipredator strategies throughout their life, but little is known about the reasons or implications of such changes. For years, it has been suggested that selection by predators should favour uniformity in local warning signals. If this is the case, we would expect high resemblance in colour across life stages in aposematic animals where young and adults share similar morphology and habitat. In this study, we used shield bugs (Hemiptera: Pentatomoidea) to test whether colour and colour diversity evolve similarly at different life stages. Since many of these bugs are considered to be aposematic, we also combined multi-species analyses with predation experiments on the cotton harlequin bug to test whether there is evidence of selection for uniformity in colour across life stages. Overall, we show that the diversity of colours used by both life stages is comparable, but adults are more cryptic than nymphs. We also demonstrate that nymphs and adults of the same species do not tend to look alike. Experiments on our model system suggest that predators can generalise among life stages that look different, and exhibit strong neophobia. Altogether, our results show no evidence of selection favouring colour similarity between adults and nymphs in this speciose clade.

From cryptic to colorful: Evolutionary decoupling of larval and adult color in butterflies.

Many animals undergo complete metamorphosis, where larval forms change abruptly in adulthood. Color change during ontogeny is common, but there is little understanding of evolutionary patterns in these changes. Here, we use data on larval and adult color for 246 butterfly species (61% of all species in Australia) to test whether the evolution of color is coupled between life stages. We show that adults are more variable in color across species than caterpillars and that male adult color has lower phylogenetic signal. These results suggest that sexual selection is driving color diversity in male adult butterflies at a broad scale. Moreover, color similarities between species at the larval stage do not predict color similarities at the adult stage, indicating that color evolution is decoupled between young and adult forms. Most species transition from cryptic coloration as caterpillars to conspicuous coloration as adults, but even species with conspicuous caterpillars change to different conspicuous colors as adults. The use of high-contrast coloration is correlated with body size in caterpillars but not adults. Taken together, our results suggest a change in the relative importance of different selective pressures at different life stages, resulting in the evolutionary decoupling of coloration through ontogeny.