Research Article Received: 24 July 2014

Revised: 12 September 2014

Accepted article published: 8 November 2014

Published online in Wiley Online Library: 5 December 2014

(wileyonlinelibrary.com) DOI 10.1002/ps.3940

Susceptibility to sulfuryl fluoride and lack of cross-resistance to phosphine in developmental stages of the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae) Rajeswaran Jagadeesan,a,b* Manoj K Nayak,a,b Hervoika Pavic,a,b Kerri Chandraa and Patrick J Collinsa,b Abstract BACKGROUND: Our aim was to ascertain the potential of sulfuryl fluoride (SF) as an alternative fumigant to manage phosphine-resistant pests. We tested the susceptibility of all life stages of red flour beetle, Tribolium castaneum (Herbst), to SF and assessed the presence of cross-resistance to this fumigant in phosphine-resistant strains of this species. RESULTS: Analysis of dose–response data indicated that the egg was the stage most tolerant to SF under a 48 h exposure period. At LC50 , eggs were 29 times more tolerant than other immature stages and adults, and required a relatively high concentration of 48.2 mg L−1 for complete mortality. No significant differences in tolerance to SF were observed among the three larval instars, pupae and adults, and all of these stages were controlled at a low concentration of 1.32 mg L−1 . Phosphine-resistant strains did not show cross-resistance to SF. CONCLUSION: Our research concluded that the current maximum registered rate of SF, 1500 gh m−3 , is adequate to control all the post-embryonic life stages of T. castaneum over a 48 h fumigation period, but it will fail to achieve complete mortality of eggs, indicating the risk of some survival of eggs under this short exposure period. As there is no cross-resistance to SF in phosphine-resistant insects, it will play a key role in managing phosphine resistance in stored-grain insect pests. © 2014 Commonwealth of Australia. Pest Management Science © 2014 Society of Chemical Industry Keywords: stored-grain pests; eggs; phosphine; alternative fumigants; cross-resistance

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INTRODUCTION

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fumigant has been reported to be relatively weak against insect eggs.22 – 26 These data are essential for selecting concentrations and exposure periods for effective fumigation and for developing tactics to manage resistance. In addition, knowledge of baseline responses to SF in T. castaneum is a prerequisite for establishing resistance testing methods. In Australia, resistance to phosphine is a major challenge for protection of stored grain.3,22 During the last two decades, detection of strong resistance to this fumigant has been recorded in several major beetle pest species,3,23,24 including T. castaneum.25 SF is currently being used selectively in bulk-grain storages to eliminate insect pests that are strongly resistant to phosphine. It is also planned that in the near future SF will be used either in rotation



Correspondence to: Rajeswaran Jagadeesan, Ecosciences Precinct, Level 3C West, GPO Box 267, Brisbane, QLD 4001, Australia. E-mail: raj.jagadeesan@daff.qld.gov.au

a Department of Agriculture, Fisheries and Forestry, Brisbane, Qld, Australia b Plant Biosecurity Cooperative Research Centre, Bruce, ACT, Australia

www.soci.org

© 2014 Commonwealth of Australia. Pest Management Science © 2014 Society of Chemical Industry

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Phosphine is by far the most widely used fumigant internationally for disinfestation of stored grains and dry processed commodities.1,2 However, this highly valuable material is now under serious threat owing to the development of resistance in target species in many regions of the world.3 – 7 Commercially available alternatives include methyl bromide, use of which is now restricted to critical circumstances,8 and sulfuryl fluoride SO2 F2 (SF). The latter has been registered in several countries as a fumigant for grain-storage structures and mills, and for treatment of grain.9 – 15 Although efficacy of SF against moth pest species infesting stored products is well documented,16 – 18 at least for short fumigation periods, there is little information on the toxicity of this fumigant against beetle pests. Results from field trials and preliminary laboratory experiments indicate that the red flour beetle, Tribolium castaneum, is more tolerant to SF than other beetle species tested.12 – 15,19 – 21 However, there is no detailed information on susceptibility to SF of individual life stages of beetle pests. In particular, information on the efficacy of SF against the egg stage of T. castaneum is inadequate and is a priority, as this

www.soci.org or as a break fumigant with phosphine, as a tactic to combat the development of resistance to phosphine and to reduce selection for resistance to SF itself. A fundamental condition for the success of such a strategy is that phosphine resistance does not provide cross-resistance to SF. This work therefore had two aims: (1) to characterise the susceptibility of all the life stages of T. castaneum, eggs, larvae, pupae and adults, to SF using dose–mortality response assays; (2) to test for cross-resistance to SF in strongly phosphine-resistant strains of T. castaneum.

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MATERIALS AND METHODS

Two sets of experiments were performed. The first set involved testing the susceptibility of all life stages (eggs, larvae, pupae and adults) to SF using a laboratory reference strain (phosphine susceptible) of T. castaneum. The second set of experiments was undertaken to determine cross-resistance to SF using adults and eggs from three strongly phosphine-resistant strains derived from Australian field samples. 2.1 Insect strains Four strains of T. castaneum were used in this study. These included: QTC4, called Lab-S in this report, a reference laboratory strain, susceptible to phosphine and other insecticides; three field-collected strains, QTC931, QNTC106 and QNTC372, called PH-SR1 , PH-SR2 and PH-SR3 respectively in this report, which are strongly resistant to phosphine. Lab-S was derived from adults collected from a storage facility in Brisbane, south-east Queensland, in 196526 and has been maintained within the laboratory without exposure to insecticides and fumigants since that time. PH-SR1 was derived from adults collected from a central storage at Natcha, south-east Queensland, in 2000,25 and PH-SR2 and PH-SR3 were collected from farms in 2006 and 2012 near Moree and Bellata in northern New South Wales respectively. These were selected with phosphine once a year, to represent their likely exposure in the field. The selection consisted of exposing 500 adult insects (2 weeks after eclosion) to 0.25 mg L−1 of phosphine over 48 h. Surviving insects were allowed to interbreed to produce the subsequent reference cultures. All the T. castaneum strains were cultured in whole wheat flour and brewer’s yeast (20:1) and maintained at 30 ∘ C, 55% relative humidity (RH) and a 12:12 h light:dark photoperiod regime. 2.2 Preparation of individual life stages Eggs, larvae (early, middle and late instars), pupae and adults of Lab-S strain were used in the first experiment. Each life stage was produced by leaving 50 unsexed adults in a jar containing 30 g of whole wheat flour (95%) and brewer’s yeast (5%) for an oviposition period of 72 h. The parental adults were then removed from all the jars, and a subset of jars, now containing unknown numbers of 1–3 days old eggs, were fumigated. For fumigation of other life stages, harvested eggs (unknown numbers) within the remaining jars were allowed to hatch and mature for 7, 14, 21 and 28 days to produce early-, middle- and late-instar larvae and pupae respectively. Finally, adult beetles (2 weeks post-eclosion) obtained from regular laboratory cultures were used for the experiment on the adult stage.

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2.3 Fumigants SF (Profume®), containing 99.8% active ingredient and 0.2% inert substance, supplied by SA Rural, Australia, was held in a cylinder and stored at 22–23 ∘ C. As required, a small quantity of the

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gas (2 L) was dispensed into a flexi foil bag (5 L) containing an adjustable valve fitted with a silicon septum (www.srcinc.com) and sampled using gas-tight syringes. The source gas concentration was determined using a Clarus 500 gas chromatograph (PerkinElmer, Waltham, MA, USA) fitted with a thermal conductivity detector. The sample was carried by helium gas through a stainless steel column (12 m × 1.41 mm o.d.) packed with porapak mesh (15.87 mm) at a flow rate of 20 mL min−1 . Injector, oven and detector temperatures were, 200, 100 and 250 ∘ C, respectively. The molecular weight of SF was used to calculate the concentration of SF per unit volume,27 and an average of eight injection samples were used to obtain a standard reference value. Phosphine was obtained by dissolving aluminium phosphide tablets in acidified water. The phosphine gas was captured in a collecting tube, and the concentration was quantified using gas chromatography according to a procedure described previously.28 2.4 Sulfuryl fluoride susceptibility tests and analyses of data Individual life stages were placed in glass jars (100 mL) with culture medium (30 g) inside airtight desiccators (4.0–6.0 L), and required concentrations of SF were injected using an airtight syringe through a rubber septum at the top of the desiccator lid. Concentrations tested include: 2.0–100 mg L−1 against eggs, 0.06–1.5 mg L−1 against the three age groups of larvae, 0.06–3.0 mg L−1 against pupae and 0.5–1.5 mg L−1 against adults. Fumigations of all the life stages were undertaken for 48 h at 25 ∘ C and 60% RH. After fumigation, the desiccators were opened and aired inside a fume cupboard. Culture medium (20 g) was added to each jar to minimise density problems, and the insects were then kept at 30 ∘ C and 60% RH for recovery and further development. The response of adult insects to SF was recorded immediately after the mortality endpoint period of 7 days, whereas the response of other life stages was evaluated at 6 weeks from the initiation of the experiment by counting the total number of adults emerged in each treatment and control. In addition, on the appearance of adults in control jars, treated jars were examined weekly, and emerged adults were counted and removed until all survivors had emerged. During fumigation, the gas concentration within each desiccator was measured twice, 1 h and 30 h after starting the fumigation, using a Clarus 500 gas chromatograph (PerkinElmer) fitted with a flame photometric detector. The sample was carried by nitrogen through a capillary column (30 m × 0.53 mm i.d × 20 μm df ) at a flow rate of 18 mL min−1 . Injector, oven and detector temperatures were, 150, 100 and 250 ∘ C, respectively. The measurements were compared with an external standard,27 and any loss of gas was compensated for by injecting more SF to maintain the desired concentrations throughout the fumigation period. Each experiment was undertaken at three separate times (three replicates), and each replicate consisted of two jars for each test concentration, which served as biological replicates. To estimate mortality, the number of adults that emerged from control jars was compared with the number of live adults that emerged in treated jars for each of the respective life stages. These values were corrected using Abbot’s formula.29 The corrected mortality (CM) value in some of the treatments was 1.0, indicates an overdispersed model and higher variability. The LC50 and LC99.9 values were estimated from the predicted probit regression equation for each life stage, and these values were used to calculate tolerance or resistance factors for each life stage or strain relative to the most susceptible stage or strain. 2.5 Phosphine susceptibility tests and data analyses Phosphine susceptibility in adults of Lab-S and the three strongly resistant strains PH-SR1 , PH-SR2 and PH-SR3 was assessed by exposing them to phosphine in gas-tight desiccators for 48 h at 25 ∘ C and 60% RH, following methods previously described.33 Mortality was counted after the endpoint period of 14 days after completion of the exposure period. Dose–mortality values at a range of concentrations of phosphine were used to estimate LC50 and LC99.9 . Resistance factors for these strains were calculated using Lab-S as the reference strain, with LC50 values and compared using a resistance ratio test.34 To bioassay eggs, groups of eggs, 1–3 days old, were harvested from Lab-S and the three strongly resistant strains PH-SR1 , PH-SR2 and PH-SR3 , as described previously in Section 2.2. Jars containing eggs were placed inside the airtight desiccators (4.0–6.0 L) and fumigated with a concentration of 0.25 mg L−1 for 48 h. This dosage is known reliably to result in complete mortality of all the life stages of Lab-S insects, including eggs (Collins PJ and Daglish GJ, unpublished), and allows survivors in PH-R1 , PH-R2 and PH-R3 , confirming the level of resistance. Phosphine was applied using an airtight syringe through a rubber septum at the top of the desiccator lid, and the concentration was measured using a gas chromatograph fitted with a thermal conductivity detector and using nitrogen as the standard.35 The experiment was repeated twice (three replicates), with each experiment consisting of two biological replicates. The responses were recorded 6 weeks after setting up the experiment. The response of eggs to phosphine was tested by analysing the percentage inhibition of egg hatch in treated jars compared with eggs hatched in control for each strain, using a one-way analysis of variance (ANOVA). Means were separated by Fisher’s protected least significant difference test at 0.05%.

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using the resistance ratio test.34 The CIs for each strain were obtained from the estimates of intercept (a), slope (b), standard error, variance and covariance, as an addition to probit analysis using Genstat.32 2.6.2 Eggs The estimated LC99.9 value of SF for eggs of Lab-S strain was used to test the response of eggs of phosphine-resistant, PH-R1 , PH-R2 and PH-R2 strains along with Lab-S strain to confirm the presence or absence of cross-resistance to SF. For each strain, eggs were harvested from 50 unsexed parental adults, as described in Section 2.2, and fumigated with the required concentration (LC99.9 ). The fumigation was performed as explained in Section 2.4, and the experiment was replicated twice (total of three replicates), with each chronological replicate consisting of two biological replicates. The responses were recorded 6 weeks after setting up the experiment. Percentage inhibition of egg hatch compared with the untreated control was calculated and analysed using a one-way ANOVA for significance. Means were separated by Fisher’s protected least significant difference test at 0.05%. These results were compared with the response of eggs to phosphine in phosphine-susceptible and strongly resistant strains.

3

RESULTS

3.1 Efficacy of SF against different life stages There was a linear response to SF in all life stages with a significant mean deviance ratio (P

Susceptibility to sulfuryl fluoride and lack of cross-resistance to phosphine in developmental stages of the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae).

Our aim was to ascertain the potential of sulfuryl fluoride (SF) as an alternative fumigant to manage phosphine-resistant pests. We tested the suscept...
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