Response and genetic analysis of malathion-specific resistant Tribolium castaneum (Herbst) in relation to population density

Extensive use of malathion for pest control on stored cereals has resulted in worldwide resistance in red flour beetles, Tribolium castaneum. In this paper we investigate population density effects on the malathion-specific resistance in PRm, a strain from the Philippines, in an integrated resistance management framework. Two populations of malathion-specific resistant (PRm) and a malathion-susceptible strain of T. castaneum were bred at different densities: low (4 adults/g) and high (12 adults/g) density on wheat plus brewer's yeast in the laboratory. After eight generations, slopes of probit regression lines and LC₅₀ values were used to monitor the effect of insect rearing density on the progression of malathion-specific resistance. The LC₅₀ of the malathion-susceptible strain (Asm) did not change significantly during selection while LC₅₀s varied for both the high-density and low-density lines of PRm, the LC₅₀ of malathion ranged from 27.51 to 34.06 and from 21.14 to 29.39 μg malathion cm⁻² for high and low density, respectively.More than 33 generations were required to achieve a 10-fold increase of resistance for the low-density line compared to only 17 generations for the high-density line. Calculations from published formulae suggested that the malathion-specific resistance of both high- and low-density lines was under monofactorial control, with complete dominance.The data showed that environmental factors such as population density differences in insect rearing and development may influence the heritability of resistance. Furthermore, the variability in results published worldwide on resistance emphasises the need to standardize test conditions across laboratories.     

Effects of phosphine and methyl bromide fumigation on the volatile flavor profile and sensory quality of dry cured ham

In separate experiments, randomized complete block designs with three replications were utilized to evaluate the effects of phosphine (PH₃) (0, 200 and 1000 ppm for 48 h) and methyl bromide (MB) (0, 4, 8, 16, and 32 mg/L for 48 h) fumigation concentration on the volatile flavor compound concentrations in dry cured ham. Minimal differences existed (P > 0.05) in the presence and concentration of aroma active compounds in both PH₃ and MB fumigated hams but sulfur and oxidation compounds were more prevalent (P < 0.05) in the fumigated treatments when compared to the control. As phosphine fumigation concentration increased, the residual concentration of phosphine also increased in the hams (P < 0.05), but all samples contained levels that are lower than the legal limit of phosphine allowed in stored food products (0.01 ppm) in the United States. A triangle test (n = 56) indicated that consumers could not discriminate (P > 0.75) between the control hams and those that were fumigated with PH₃. Minimal aroma/flavor differences existed among MB, PH3 and control hams, and dry cured ham that was fumigated with PH₃ was safe for consumption based on residual phosphine concentrations in the meat tissue.     

Optimisation of synthesis of oligosaccharides derived from lactulose (fructosyl-galacto-oligosaccharides) with β-galactosidases of different origin

Batch synthesis of fructosyl-galacto-oligosaccharides from lactulose was performed with commercial β-galactosidase preparations from Aspergillus oryzae, Kluyveromyces lactis and Bacillus circulans. The enzyme from A. oryzae produced the highest yield and specific productivity of synthesis, being selected for further studies. Optimization of fructosyl-galacto-oligosaccharides synthesis was carried out using response surface methodology, considering temperature and initial sugar concentration as variables and yield and specific productivity as response parameters. Maximum yield of 0.41 g g⁻¹ fructosyl-galacto-oligosaccharides was obtained at 70 °C and 60% w/w lactulose concentration, while maximum specific productivity of 1.2 g h⁻¹ mg⁻¹ was obtained at 70 °C and 40% w/w lactulose concentration.     

Production of d-galactose using β-galactosidase and Saccharomyces cerevisiae entrapped in poly(vinylalcohol) hydrogel

d-Galactose was produced from lactose (200 g l⁻¹) in the batch mode of a simultaneous saccharification and fermentation process (SSF). β-Galactosidases (from Kluyveromyces lactis and Aspergillus oryzae) and yeasts were immobilized in poly(vinylalcohol) hydrogel lens-shaped capsules – LentiKats^®. After 20 repeated batch runs with entrapped K. lactis, β-galactosidase and free Saccharomyces cerevisiae (10% v/v inoculum), galactose productivity decreased to 50% and 1.4 kg of galactose were prepared. Compared to this, just 20% decrease of galactose productivity and a 0.9 kg production of galactose were observed for the SSF process with β-galactosidase from A. oryzae after 15 repeated batches under the same conditions. In the process of SSF with co-immobilized enzyme from K.lactis and S.cerevisiae, the galactose productivity increased from 3 g l⁻¹ h⁻¹ to 4.1 g l⁻¹ h⁻¹, which reduced the time of preparation of d-galactose.     

Effect of modifiers for As,Cu and Pb determinations in sugar-cane spirits by GF AAS

Palladium plus magnesium nitrates with and without Ir, Ru and W were evaluated for the simultaneous determination of As, Cu and Pb in cachaça by graphite furnace atomic absorption spectrometry. For 20 μL of sample, 5 μL Pd(NO₃)₂ and 3 μL Mg(NO₃)₂ dispensed together onto the Ir-coated platform of the THGA, analytical curves in the 0–30.0 μg L⁻¹ As, 0–1.50 mg L⁻¹ Cu and 0–60.0 μg L⁻¹ Pb were built up and typical linear correlation coefficients were always better than 0.999. The limit of detection was 1.30 μg L⁻¹ As, 140 μg L⁻¹ Cu and 0.90 μg L⁻¹ Pb. As, Cu and Pb contents in 10 cachaça samples agreed with those obtained by ICP-MS. Recoveries of spiked samples varied from 96% to 106% (As), 97% to 112% (Cu) and 92% to 108% (Pb). The relative standard deviation (n = 12) was typically 2.7%, 3.3% and 1.9%.     

Development of polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for sodium saccharin residue in food samples

A sensitive and specific polyclonal antibody (PcAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) for sodium saccharin is described. 6-Amino saccharin was coupled to carrier protein for artificial antigen by diazotisation. New Zealand white rabbits were immunised to obtain anti-sodium saccharin PcAb and then icELISA was developed. The assay showed high sensitivity and specificity to sodium saccharin, with the 50% inhibition value (IC₅₀) of 0.243 μg mL⁻¹, workable range (IC₃₀–IC₇₀) of 0.050–12.8 μg mL⁻¹ and limit of detection (LOD, IC₂₀) of 0.021 μg mL⁻¹. The average recoveries of sodium saccharin in spiked food samples were estimated ranging from 70.7% to 98.8%. A statistically significant correlation of results was obtained between this new ELISA and previously established HPLC approaches with the food-relevant sodium saccharin concentration range 0–320 μg mL⁻¹ (R² = 0.9887–0.9975). These results indicated that the established ELISA was a potential and useful analytical tool for rapid determination of sodium saccharin residue in food samples.     

Phosphine resistance in Brazilian populations of Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae)

Phosphine resistance was assessed in adults of 22 Brazilian populations of Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae). The concentration-mortality bioassays for the detection of phosphine resistance followed the FAO standard method. Twenty populations of S. zeamais were resistant to phosphine and the resistance ratios at the LC₅₀ ranged from 1.1- to 86.6-fold. This is the first report of phosphine resistance in populations of S. zeamais in Brazil, where previous surveys did not detect resistance in this species. There was significant variation in respiration rate (CO₂ production) among the populations (P < 0.05). Respiration rate was significantly inversely correlated with phosphine resistance for this species (P < 0.05). The populations with lower respiration rates showed higher levels of phosphine resistance, suggesting that the lower respiration rate is associated with the physiological basis of phosphine resistance due to reduced fumigant uptake.     

A rapid assay for the detection of resistance to phosphine in the lesser grain borer,Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae)

Resistance to the fumigant phosphine in stored product insect pests is a global problem. Diagnosis of resistance relies on a bioassay developed by the FAO that involves a mortality assessment after 20-h fumigation of a pest population at a discriminating concentration of gas, followed by a 14-day post fumigation assessment. This bioassay is impractical for monitoring and early detection of phosphine resistance in routine pest management. We utilized the procedure of a commercial resistance detection test kit for rapid detection in field populations of lesser grain borer, Rhyzopertha dominica (F.). We established a knockdown effect of either susceptible or resistant insects by exposing them to a high concentration of phosphine. We assessed the relationship between adult knockdown times and the FAO method for 18 beetle populations utilizing knockdown criteria for a single beetle in a chamber, or for 50% or 100% knockdown times for groups of beetles, exposed to 3000 ppm of phosphine. We also determined the most effective concentrations that would elicit the quickest knockdown while estimating the recovery times from exposure. Results suggest that a KT₁₀₀ test was better than the KT₅₀ and the KT_single tests. Based on the responses of susceptible populations, we established that a KT₁₀₀ of approximately 18 min can be used as a viable knockdown time to distinguish a susceptible from a resistant populations. Higher concentrations of phosphine significantly elicited a quicker recovery in strongly resistant populations compared to susceptible populations. These findings have potential for developing a robust commercial kit for practical phosphine resistance detection in populations of R. dominica by commercial fumigators, and could be incorporated in a resistance management program.     

Chitosan-tethered the silica particle from a layer-by-layer approach for pectinase immobilization

“Spherical polyelectrolyte brush” of core–shell structure were prepared by grafting poly (sodium 4-styrenesulphonate) (PSStNa) from SiO₂ nanoparticle via the surface-initiated atom transfer radical polymerization strategy. The colloidal stability was not impeded by the adsorbed proteins despite the fact that up to 316.8 mg of enzyme was adsorbed per gram of the carrier particles. The immobilized pectinase revealed acceptable pH stability over a broad experimental range of 3.0–4.5. The activity half lives for native and bound states of enzyme were found as 13.5 d and 30 d, respectively. The activity of immobilized pectinase adsorbed onto these particles was analyzed in terms of the Michaelis–Menten parameters. Kinetic parameters were calculated as 8.28 and 9.98 g pectin ml⁻¹ for K_m and 1.165 × 10⁻³ g pectin s⁻¹ g enzyme⁻¹ 1.124 × 10⁻³ g pectin s⁻¹ g particle⁻¹ for V_max in the case of free and immobilized enzymes, respectively. Enzyme activity was found to be approximately 49.7% for immobilized enzyme after storage for 1 month.     

Trace element levels in raw milk from northern and southern regions of Croatia

A total of 157 raw milk samples were collected from tankers arriving at processing facilities from rural areas in northern and southern regions of Croatia during 2009 and 2010. Concentrations of As, Cd, Cu, Hg and Pb in the samples were analysed by graphite furnace-atomic absorption spectroscopy. Mean Pb concentrations in northern and southern regions were 58.7 and 36.2 μg l⁻¹, respectively, and both exceeded the maximum recommended level. Arsenic concentrations ranged from 1 to 283 μg l⁻¹ in the southern and to 1019 μg l⁻¹ in the northern regions. Mean Cd and Hg levels were: 1.76 and 1.59 in the northern and 3.4 and 7.1 μg l⁻¹ in the southern region. Significantly higher Cd and Hg levels were observed in the southern than in the northern region (p < 0.001, both). Similar mean Cu levels were found in both regions: 931.9 in the north and 848.4 μg l⁻¹ in the south. The results indicate that particular attention should be paid to Pb residues. In future studies, a greater number of milk samples and grass samples from pastures from different regions of Croatia should be controlled to confirm the absence of possible toxicological risks.     

Pico molar assay of riboflavin in human urine using voltammetry

Using a new type of DNA and carbon nano tube (CNT) mixed paste electrode using cyclic and square wave anodic stripping voltammetric (SWASV) methods, this study presents an assay of riboflavin (RF) under optimum conditions. Results of the experiment yielded a low working concentration range of nanograms with 1–10 and 10–170 ng L⁻¹ and 5–105 μg L⁻¹, at an accumulation time of 80 s in a 0.1 M H₃PO₄ electrolyte solution. A relative standard deviation of 30 μg L⁻¹ was observed at an accuracy level of 0.1164% (n = 15) under optimum conditions. The detection limit (S/N) was pegged at 0.2 ng L⁻¹ (5.31 × 10⁻¹³ mol L⁻¹ RF). The proposed method was successfully applied to an actual human urine and drug sample, and can be applied to assays of other biological samples.     

Phosphine resistance in saw-toothed grain beetle,Oryzaephilus surinamensis in the United States

Phosphine (PH₃) fumigation resulting in sub-lethal exposure has led to the development of phosphine resistance in many stored-product insect species worldwide and is a major challenge to the continued effective use of phosphine. In 2016 phosphine resistance was found in Tribolium castaneum (Herbst) and Plodia interpunctella (Hübner) collected from California dried fruit and nut processing facilities. Although Oryzaephilus surinamensis (L.) infests grain, dried fruit, and nuts in storage and processing facilities, phosphine resistance in this species has not been studied in the United States. In this study, the discriminating dose of phosphine for O. surinamensis eggs was estimated using a laboratory susceptible strain; it was found to be 28.4 ppm over a 72-h fumigation period (1 mg/L of phosphine = 714.18 ppm or 1 ppm = 0.0014 mg/L). Discriminating dose bioassays were used to determine phosphine resistance in both eggs and adults of 14 different populations collected from California and Oklahoma. Resistance to phosphine was detected in four out of 14 populations in adults and nine out of 14 populations in eggs and ranged from 2 to 100%. Phosphine percent survival values in both adults and eggs of three populations, namely, Box BR, Box BF, and OKWat were >90%. Lethal concentration values required to kill 99% of individuals in samples for adults of these three populations were predicted as 320.5, 290.7, and 263 ppm, respectively, and those for eggs were 1030.7, 1055.9, and 564.5 ppm, respectively, over a 72-h fumigation period. This study confirms that phosphine resistance is present in O. surinamensis in the United States.     

Selective determination of copper and iron in various food samples by the solid phase extraction

The solid phase extraction method developed using N-benzoyl-N-phenylhydroxylamine as a chelating reagent and Amberlite XAD-1180 as an adsorbent was used for the determination of Cu(II) and Fe(III) in various food samples by flame atomic absorption spectrometry. The samples were digested by using nitric acid and hydrogen peroxide. The Cu concentrations ranged from 1.01 to 5.81 μg g⁻¹ in cereals, from 0.40 to 9.67 μg g⁻¹ in vegetable and fruits and from 0.37 to 0.70 μg g⁻¹ in infusions while the Fe concentrations ranged from 7.48 to 34.3 μg g⁻¹ in cereals, from 5.74 to 260 μg g⁻¹ in vegetable and fruits, from 1.63 to 5.12 μg g⁻¹ in infusions and from 0.24 to 1.56 mg L⁻¹ in beverage samples. The Cu and Fe concentrations found were compared with the results obtained from the other food studies in the world.     

Effect of processing and storage of Jameed on conjugated linoleic acid content and fat and cholesterol oxidation

The influence of Jameed processing and storage on fat and cholesterol oxidation, hydrolytic rancidity, and conjugated linoleic acid (CLA) was evaluated by determining peroxide value (PV), 7-ketocholesterol, free fatty acids content (FFAs) and total CLA. Three different sizes of Jameed pieces (ca. 50, 200 and 400 g balls) were used to investigate the influence of storage for 0, 1, 3, 5 and 7 months on the above lipid oxidation parameters. No significant (P>0.05) effect of the Jameed processing steps on lipid changes was found with the exception of the sun drying steps, that caused an increase of PV, 7-ketocholesterol and FFA contents and a decrease in CLA content. This trend was also observed during storage of Jameed at room temperature and kept exposed to light and atmospheric air. The PV values of the 50, 200 and 400 g balls after 7 months of storage were 35.3, 19.4 and 20.4 meq O₂ kg⁻¹ of fat, that were 17, 11 and 12 times higher compared with the corresponding values of the fresh samples (1.9 meq O₂ kg⁻¹ of fat ). The 7-ketocholesterol of the tested samples increased from about 5.7 μg g⁻¹ to 210, 120 and 125 μg g⁻¹ for the 50, 200 and 400 g balls, respectively. FFA content increased remarkably during storage, from about 1.3 g kg⁻¹ at the beginning of storage to 8.6 g kg⁻¹ of fat after 7 months with no significant (P>0.05) effect for the size of Jameed pieces on FFAs content. On the contrary, a decrease of about 43%, 26% and 26% of the original value (5.7 mg kg⁻¹ of fat) in CLA content was found for the stored Jameed pieces of 50, 200 and 400 g, respectively.     

Genetic diversity and its geographic structure in Sitophilus oryzae (Coleoptera; Curculionidae) across India – implications for managing phosphine resistance

The rice weevil, Sitophilus oryzae, is a serious global pest of stored grains. Fumigation with phosphine gas is the primary control method for S. oryzae, but the indiscriminate and prolonged use of phosphine gas has led to the development of heritable resistance. Developing and implementing an effective phosphine resistance management strategy for S. oryzae relies on an understanding of its genetic diversity and any structuring of that diversity geographically. We therefore sequenced the mitochondrial cytochrome c oxidase subunit I gene from 143 S. oryzae specimens collected from 37 locations across India, and from that assessed the genetic diversity of the species and its phylogeographic structuring. In addition, we compared the genetic diversity in Indian S. oryzae populations (the hypothesised origin of this beetle) to global populations. Genetic diversity was low in Indian S. oryzae, with only eight haplotypes (including two very common haplotypes) identified. The low level of mitochondrial diversity observed in this species appears typical of stored product pests, perhaps suggesting that low mitochondrial diversity is associated with repeated phosphine fumigations, which may eliminate low frequency haplotypes. The genetic diversity of S. oryzae in India is, however, higher than in many other countries, though comparable levels were identified in China. There was no evidence of population genetic structure across India, with most haplotypes found in three of the broad biogeographic regions. This lack of phylogeographic structuring indicates significant gene-flow across India, most likely through the incidental anthropogenic transport of this relatively poor (or reluctant) flyer. The major practical implication is that phosphine resistance management for S. oryzae needs to be dealt with country wide, as populations are not isolated.     

Determination of pyrethroids in porcine tissues by matrix solid-phase dispersion extraction and high-performance liquid chromatography

An effective matrix solid-phase dispersion (MSPD) extraction for determination of two pyrethroids (cypermethrin and deltamethrin) in porcine tissues (liver, muscle, heart and kidney) is described. A neutral alumina-based MPSD column was used for extraction of analytes. The high-performance liquid chromatography and ultraviolet detector was applied using a reverse-phase C₁₈ column and acetonitrile-water (85:15, v/v) was used as mobile phase. The good linear fit curve ranging from 0.05 to 50 μg mL⁻¹ for cypermethrin (CM) and deltamethrin (DM) was obtained with a regression coefficient (r) of 0.999. Recoveries at 0.2 and 0.5 μg g⁻¹ levels were between 83.5% and 109%. The limits of detection and quantification were: 0.01 and 0.026 μg g⁻¹ for CM, 0.017 and 0.056 μg g⁻¹ for DM, respectively. The proposed method was successfully applied to the determination of the pyrethroids in porcine tissues.     

Scrubbing carbon dioxide prevents overestimation of insect mortality in long-duration static phosphine toxicity assays

Exposing insects to toxicants such as phosphine (PH₃) in sealed chambers is a common procedure in fumigant efficacy testing. During long exposures, carbon dioxide (CO₂) produced by metabolic processes of fed insects may accumulate in the vessel. As CO₂ enhances the toxicity of several fumigants, concomitant exposure of fumigant and CO₂ may lead to erroneous measurements of insect mortality and thus fumigant toxicity. In this study, the effect of a CO₂ scrubber such as soda lime (a solid formulation of wet hydroxides of calcium, sodium and potassium) or periodic flushing of the headspace, on insect mortality in static PH₃ toxicity assays was compared with an “unscrubbed” (no intervention) treatment. Soda lime was highly effective in removing CO₂ from the headspace of sealed chambers, without measurable loss of PH₃. Mortality of Sitophilus oryzae (L.) adults treated with phosphine in chambers scrubbed with soda lime or flushed (opened) daily was considerably lower than in unscrubbed chambers. Twenty grams of soda lime per litre of exposure chamber is recommended to obtain accurate static fumigant mortality results.     

Tungsten permanent chemical modifier with co-injection of Pd(NO3)2 + Mg(NO3)2 for direct determination of Pb in vinegar by graphite furnace atomic absorption spectrometry

A tungsten carbide coating on the integrated platform of a transversely heated graphite atomizer (THGA^®) used together with Pd(NO₃)₂ + Mg(NO₃)₂ as modifier is proposed for the direct determination of lead in vinegar by graphite furnace atomic absorption spectrometry. The optimized heating program (temperature, ramp time, hold time) of atomizer involved drying stage (110 °C, 5 s, 30 s; 130 °C, 5 s, 30 s), pyrolysis stage (1000 °C, 15 s, 30 s), atomization stage (1800 °C, 0 s, 5 s) and clean-out stage (2450 °C, 1 s, 3 s). For 10 μL of vinegar delivered into the atomizer and calibration using working standard solutions (2.5–20.0 μg L⁻¹ Pb) in 0.2% (v/v) HNO₃, analytical curve with good linear correlation (r = 0.9992) was established. The characteristic mass was 40 pg Pb and the lifetime of the tube was around 730 firings. The limit of detection (LOD) was 0.4 μg L⁻¹ and the relative standard deviations (n = 12) were typically <8% for a sample containing 25 μg L⁻¹ Pb. Accuracy of the proposed method was checked after direct analysis of 23 vinegar samples. A paired t-test showed that results were in agreement at 95% confidence level with those obtained for acid-digested vinegar samples. The Pb levels varied from 2.8 to 32.4 μg L⁻¹. Accuracy was also checked by means of addition/recovery tests and recovered values varied from 90% to 110%. Additionally, two certified reference materials were analyzed and results were in agreement with certified values at a 95% confidence level.     

Analysis of multi-pesticide residues in the foods of animal origin by GC–MS coupled with accelerated solvent extraction and gel permeation chromatography cleanup

A new analytical method was developed to simultaneously determine residues of 109 pesticides (including isomers) in the foods of animal origin. Acetonitrile was selected for accelerated solvent extraction (ASE) for effectively extracting the pesticides from the fatty samples. The cleanup was performed with an automated gel permeation chromatography (GPC) cleanup system. The prepared samples were analysed with GC–MS in the selected ion monitoring mode (SIM) using one target and two qualitative ions for each analyte. Chlorpyrifos-d₁₀ was used as an internal standard. The lowest limit of detection was 0.3 μg kg⁻¹ for some pesticides. The recoveries and relative standard deviations (RSDs) were checked by spiking untreated samples with pesticides at 0.05, 0.1 and 0.2 mg kg⁻¹. The average recoveries of most pesticides were from 62.6% to 107.8%. The precision values expressed as RSD were all ?20.5% (n = 6). Good linearity (r ? 0.99) was observed between 0.05 and 1.5 μg mL⁻¹.     

Impact of resistance on the efficacy of binary combinations of spinosad, chlorpyrifos-methyl and s-methoprene against five stored-grain beetles

Laboratory experiments were conducted to determine the efficacy of spinosad (a biopesticide), chlorpyrifos-methyl (an organophosphorus compound (OP)) and s-methoprene (a juvenile hormone analogue) applied alone and in binary combinations against five stored-grain beetles in wheat. There were three strains of Rhyzopertha dominica, and one strain each of Sitophilus oryzae, Tribolium castaneum, Oryzaephilus surinamensis and Cryptolestes ferrugineus. These strains were chosen to represent a range of possible resistant genotypes, exhibiting resistance to organophosphates, pyrethroids or methoprene. Treatments were applied at rates that are registered or likely to be registered in Australia. Adults were exposed to freshly treated wheat for 2 weeks, and the effects of treatments on mortality and reproduction were determined. No single protectant or protectant combination controlled all insect strains, based on the criterion of >99% reduction in the number of live F₁ adults relative to the control. The most effective combinations were spinosad at 1 mg kg⁻¹+chlorpyrifos-methyl at 10 mg kg⁻¹ which controlled all strains except for OP-resistant O. surinamensis, and chlorpyrifos-methyl at 10 mg kg⁻¹+s-methoprene at 0.6 mg kg⁻¹ which controlled all strains except for methoprene-resistant R. dominica. The results of this study demonstrate the difficulty in Australia, and potentially other countries which use protectants, of finding protectant treatments to control a broad range of pest species in the face of resistance development.