Development and Validation of a Quick Easily Used Biochemical Assay for Evaluating the Viability of Small Immobile Arthropods

Phillips, C.B.; Iline, I.I.; Richards, N.K.; Novoselov, M. A.; McNeill, M.R.

doi:10.1603/ec13028

Cited by 12 publications

(23 citation statements)

References 42 publications

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“…The next main hurdle will either be to develop ways of reducing persistent postmortem activity of enzymes in solution or to streamline and simplify the electrophoretic procedure so it is more suitable for adoption by pest managers. Our method for producing histochemical reactions in microtubes from crude macerates of small samples holds promise for developing a variety of quick, easily used assays for use outside of the laboratory (e.g., Iline et al 2013;Novoselov et al 2013;Phillips et al 2013).…”

Section: Discussionmentioning

confidence: 99%

“…We first evaluate postmortem enzyme degradation using polyacrylamide gel electrophoresis, then by assaying enzymes in solution. Our method for assaying enzymes in solution is particularly amenable to developing, quick easily used tests suited to use outside the laboratory for a range of applications Novoselov et al 2013;Phillips et al 2013). …”

Section: Introductionmentioning

confidence: 99%

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Potential to exploit postmortem enzyme degradation for evaluating arthropod viability

et al. 2014

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Inspecting for live organisms is the main method used to verify efficacy of phytosanitary treatments. Evaluating whether small, immobile organisms such as eggs, pupae and scale insects are alive or dead usually involves either checking morphological criteria or rearing them to observe development. These methods can be inaccurate, impractical and time consuming; thus, better methods are needed. To evaluate the potential for developing enzyme-based viability assays, we used electrophoretic gels to evaluate postmortem degradation of ten enzymes in Musca domestica L. (Diptera: Muscidae), four in Bemisia flocculosa Gill and Holder (Hemiptera: Aleyrodidae), and seven in Listronotus bonariensis (Kuschel) (Coleoptera: Curculionidae). Fresh insects displayed strong enzyme activity and distinct bands, but dead insects exhibited either no activity or weakened activity with reduced band resolution and increased migration of stained areas. Of ten enzymes investigated, seven showed clear indications of degradation just 1 day postmortem. Polyacrylamide gel electrophoresis of enzymes can be used to evaluate organism viability and has potential for estimating postmortem intervals. We also measured postmortem degradation rates of five M. domestica enzymes by assaying them in solution; these showed constant or gradually declining activity for 28 days postmortem, so live and dead specimens were less easily distinguished. By assaying enzymes in solution, it is possible to develop quick, easily operated tests that can be used outside the laboratory for a variety of quarantine-related purposes.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Potential to exploit postmortem enzyme degradation for evaluating arthropod viability

et al. 2014

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“…Exceptions were activity stains for coniferyl and cinnamyl dehydrogenases, which used coniferyl alcohol or cinnamyl alcohol (Halpin et al 1992;Sauter & Kende 1992;Pandey et al 2011). To most recipes, 0.1% Triton X-100 was added, and NBT was replaced with MTT and PMS with mPMS (Phillips et al 2013(Phillips et al , 2014. Results were evaluated after staining for 30 min.…”

Section: Activity Staining Of Enzymesmentioning

confidence: 99%

“…Preliminary tests indicated that glucose was present only in low concentrations in these samples so, for brevity, the latter two assays are referred to as the 'fructose test' and the 'sucrose test' . Recipes were modified by adding 0.1% Triton X-100 and replacing NBT with MTT and PMS with mPMS (Phillips et al 2013(Phillips et al , 2014. Results were evaluated after staining for 5 min.…”

Section: Evaluation Of Sugar Concentrationmentioning

confidence: 99%

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Towards a test to verify that wood has been heattreated to the ISPM15 standard

Iline¹,

Novoselov²,

Richards³

et al. 2014

NZPP

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International trade of wood including dunnage is a wellknown pathway for spreading diseases and woodboring insects between countries To mitigate this risk the International Standard for Phytosanitary Measures No 15 (ISPM15) states that wood to confirm that wood has been properly heattreated After preliminary investigations of 12 enzymes and 3 sugars from pine xylem experiments focused on malate dehydrogenase (MDH) and fructose Samples from the surfaces of heattreated Pinus radiata wood exhibited decreased MDH activity and increased fructose concentration However samples from 5 mm deeper in the profile of heated wood showed similar MDH activity to unheated wood but contained relatively lower fructose concentrations There is potential to exploit heatinduced changes in MDH activity and fructose concentration to develop a quick easilyused assay for verifying compliance of wood packaging materials with ISPM15

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Methyl bromide fumigation and delayed mortality: safe trade of live pests?

et al. 2014

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Live organisms intercepted from treated commodities during phytosanitary inspections usually arouse suspicions of treatment failure, sub-standard treatment application, or post-treatment infestation. The additional possibility that some treatments could kill slowly, meaning commodities might be inspected before pests have succumbed, is seldom considered for treatments other than irradiation. We used a novel biochemical viability assay to measure delays between methyl bromide fumigation and mortality of dipteran eggs, and evaluated the correspondence between egg viability and egg morphological features. Our experimental conditions simulated shipping of rock melons from Australia to New Zealand by sea and air. No eggs survived fumigation, but they took 3–20 days to die, whereas phytosanitary inspections of rock melons occur within 2–7 days. Delays were not influenced by methyl bromide concentration, but were significantly lengthened by cooler storage temperatures. Methyl bromide’s preservative effects delayed degradation of egg morphology, so the biochemical assay detected mortality long before morphological signs of egg death appeared. The results show that commodities subjected to effective methyl bromide treatments are at risk of being inspected before all pests have either died, or started to exhibit morphological signs of death. This could cause commodities to be unnecessarily rejected by quarantine authorities. Better methods than inspection for live pests are needed to assist authorities to gain assurance that treated commodities have been effectively disinfested. These could be developed by exploiting biochemical responses of pests and commodities to treatments.

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Development and Validation of a Quick Easily Used Biochemical Assay for Evaluating the Viability of Small Immobile Arthropods

Cited by 12 publications

References 42 publications

Potential to exploit postmortem enzyme degradation for evaluating arthropod viability

Potential to exploit postmortem enzyme degradation for evaluating arthropod viability

Towards a test to verify that wood has been heattreated to the ISPM15 standard

Methyl bromide fumigation and delayed mortality: safe trade of live pests?

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