Dataset Information

High-quality DNA isolation protocol for detection of Khapra beetle (Dermestidae: Trogoderma granarium Everts, 1898) in standard wheat germ trap.

ABSTRACT:

Background

Khapra beetle (Dermestidae: Trogoderma granarium Everts, 1898) is an internationally significant pest of grain crops and stored grain products. Wheat germ traps, routinely used in surveillance sampling of Khapra beetle provide feed-substrates used by the pest throughout its life cycle. However, Khapra beetle larvae, eggs and other traces of the pest, such as larval frass and exuviae, in wheat germ traps are difficult to sort and taxonomically identify. Additionally, high levels of polysaccharides in wheat germ can inhibit PCR based molecular detection of this pest captured in the traps.

Methods and results

We have developed a sensitive and low-cost protocol for extracting trace levels of Khapra beetle DNA from an entire wheat germ trap. Overnight digestion of entire trap contents in 6 mL of ATL buffer, followed by a 40 min lysis step was optimal for DNA extraction. Paired with reported qPCR assays, this protocol allows the detection of a few hairs of T. granarium in a typical 2-gram wheat germ trap.

Conclusion

This DNA extraction protocol makes it possible to perform a more rapid identification of the pest following wheat germ sample collection. The protocol has potential to improve international efforts for Khapra beetle surveillance.

SUBMITTER: Zhu X

PROVIDER: S-EPMC10520210 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Publications

High-quality DNA isolation protocol for detection of Khapra beetle (Dermestidae: Trogoderma granarium Everts, 1898) in standard wheat germ trap.

Zhu Xiaocheng X Gopurenko David D Galea Francesca F Marsh Ian B IB McDougall Sandra S Thotagamuwa Agasthya A

Molecular biology reports 20230804 10

<h4>Background</h4>Khapra beetle (Dermestidae: Trogoderma granarium Everts, 1898) is an internationally significant pest of grain crops and stored grain products. Wheat germ traps, routinely used in surveillance sampling of Khapra beetle provide feed-substrates used by the pest throughout its life cycle. However, Khapra beetle larvae, eggs and other traces of the pest, such as larval frass and exuviae, in wheat germ traps are difficult to sort and taxonomically identify. Additionally, high level ...[more]

PMID: 37541998

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Project description:BackgroundKhapra beetle (Trogoderma granarium Everts) is a significant pest of food products around the world, causing great losses of stored grain and produce, with export restrictions imposed on countries with established beetle populations. Khapra beetle is a high-priority exotic invertebrate pest in many countries requiring a rapid quarantine/biosecurity response when incursions occur. To address this, we developed a novel Khapra LAMP (loop-mediated isothermal amplification) assay using a portable real-time fluorometer and an additional 18S ribosomal DNA (18S) insect control LAMP assay for confirmation of the presence of insect DNA. Both LAMP tests can be performed either in a portable real-time fluorometer or using simple, visual colorimetric technique.ResultsBoth the Khapra and 18S LAMP tests amplify positive samples within ≤ 25 min, with an anneal derivative temperature of 77.7 ± 0.7 °C for Khapra LAMP test and 88.0 ± 1.0 °C for 18S. The new Khapra LAMP assay is sensitive to very low levels of DNA (1.02 × 10-6 ng μL-1 ). Additionally, we developed a gBlock double stranded DNA fragment for use as positive Khapra control with a different anneal derivative of 80 °C. Both assays are simple to use in the field and are capable of amplifying DNA from target beetles, even when samples are partially degraded which is typically found during surveillance activities. By screening a broad panel of Dermestidae species we demonstrate that our new assay is species-specific, with no detections of false positives. Also, we evaluated multiple DNA extraction methods, with both QuickExtract and HotSHOT extraction methods proving suitable for in-field use.ConclusionThe novel Khapra and 18S LAMP assays should improve speed, accuracy and confidence of detection of Khapra beetle at incursion points and aid rapid biosecurity responses in any country affected, especially as the assays described here are portable and easy to implement in the field conditions where resources are limited. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Project description:Local potential competitor species are important determinants of the invasibility of an environment even when widely recognized invasive species are concerned since it may compromise its establishment. Thus, the outcome of the direct competition among the invasive khapra beetle, Trogoderma granarium, and the cosmopolitan species lesser grain borer, Rhyzopertha dominica and rice weevil, Sitophilus oryzae, and thus the likelihood of establishment of T. granarium under their co-occurrence, was here explored in paddy rice and wheat, at temperatures between 25 and 35°C and through 200 days of storage. Insect infestations were higher in wheat rather than in paddy rice. Trogoderma granarium was unable to displace any of the competing species under two and three-species competition experiments retaining lower adult population than both local competitors at the lowest temperature level. Rhyzopertha dominica prevailed in paddy rice, while S. oryzae prevailed in wheat. Paradoxically, T. granarium adults retained low population growth but contributed more for the total frass production and grain loss, much more than that recorded for R. dominica. Nonetheless, T. granarium larvae exhibited high population numbers 130 days after the introduction of the parental individuals. At higher temperature levels (30 and 35°C) the numbers of T. granarium larvae were extremely high even after 65 days, while the numbers of the other two species rapidly declined. Interestingly, the simultaneous presence of R. dominica and S. oryzae was beneficial for the population growth of T. granarium. Consequently, T. granarium has the ability to outperform other primary stored-product insects at high temperatures, while its presence at low temperatures remains for long periods apparently unaffected by other co-occurring species. Hence, T. granarium, in wheat, is able to outcompete other major species of stored-product insects at elevated temperatures, while at 25°C this species can maintain low numbers of individuals for long periods, which can rapidly produce population outbursts when the prevailing conditions are suitable for its development.

Project description:Bag storage of wheat is common in Asian countries, and common types of such bags include jute bags, woven plastic bags, and hermetic plastic bags. In order to assess infestation by two serious pests, namely Rhyzopertha dominica (Coleoptera: Bostrichidae) and Trogoderma granarium (Coleoptera: Dermestidae) in these bags, two experiments were performed. In the first experiment, three popular wheat varieties, namely Akbar, Dilkash, and Bakhar star were filled in above-mentioned miniature-size bags which were then placed in jars with three replicates per bag type and variety. Forty insects (adults for R. dominica and larvae for T. granarium) were released in the center of the jars for a period of 30 d in two different setups for both species. Data were recorded twice: after 15 d and 30 d. Results showed insects entered the jute bags only (made invasions due to its loose fibers and openings). Inside the bags, R. dominica caused more weight loss and live insects in Dilkash and Akbar varieties, while T. granarium preferred Bakhar star and Dilkash compared with their third variety. In the second experiment, only two bags, namely woven plastic bags and hermetic plastic bags filled with one variety, were tested to check damage (penetrations because no openings are present in these bags as in jute bags) to the bags. Results showed both species created holes in woven plastic bags but not in hermetic plastic bags. These results showed hermetic plastic bags remained safe from external infestation compared with the other two bags. Sometimes if insects are already present in the grains inside the bags, fumigation is needed from outside to kill the pests inside. To evaluate this, all three bags were filled with a wheat variety and were also infested with both insect species and placed in a fumigation container with nine replicates per bag type. A phosphine tablet (3.0 g) wrapped in muslin cloth was placed in a container which was then sealed, and the fumigation-induced mortality after 24 h was recorded. Results showed mortality was >95% to 100% in woven and jute bags, respectively, while mortality in hermetic plastic bags remained very low (<3%). These results revealed the least fumigant gas permeation in hermetic plastic bags compared with jute and woven plastic bags. Results of all three experiments demand immediate alteration in creation of all three bags to curtail infestation from outside (jute and woven plastic bags) as well as to generate maximum fumigation efficacy when the source of infestation is with the grains (hermetic bags).

Project description: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.

Dataset Information

High-quality DNA isolation protocol for detection of Khapra beetle (Dermestidae: Trogoderma granarium Everts, 1898) in standard wheat germ trap.

Background

Methods and results

Conclusion

Publications

High-quality DNA isolation protocol for detection of Khapra beetle (Dermestidae: Trogoderma granarium Everts, 1898) in standard wheat germ trap.

Similar Datasets

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