Prevalence of resistance to deltamethrin in Rhyzopertha dominica (F.) in eastern Australia

The pyrethroid, deltamethrin, is currently used as a grain protectant to protect stored grain from insect pests in several countries including Australia. We examined the variation in response to wheat treated with deltamethrin among 15 field populations of the lesser grain borer, Rhyzopertha dominica (F.) collected from Queensland and New South Wales in eastern Australia during 2008 and 2009, to establish whether resistance has developed in this species. Based on the results of treated wheat bioassays, using a discriminating dose of 0.5 mg kg⁻¹, we found that 14 of the 15 field populations were resistant to deltamethrin. The distance between the two most geographically separated field populations was 450 km, and both of these populations contained resistant insects. Deltamethrin applied at 1 mg kg⁻¹ alone or with the synergist piperonyl butoxide at 8 mg kg⁻¹ failed to control resistant R. dominica. On the basis of our findings, we conclude that populations with deltamethrin resistant individuals are common in the eastern part of Australia, and control failures are expected when such populations are present in treated grain.     

Effects of habitat fragmentation on genetic diversity and population differentiation of Liposcelis bostrychophila badonnel (Psocoptera: Liposcelididae) as revealed by ISSR markers

Liposcelis bostrychophila is a globally important stored product pest that is adapted to many habitats and is difficult to control due to insecticide resistance. We evaluated effects of habitat fragmentation on the genetic diversity and differentiation of L. bostrychophila populations. These genetic data were collected in Wuhu city, China using inter-simple sequence repeat (ISSR). A total of 108 discernible DNA fragments were detected, 107 were polymorphic and the percentage of polymorphic bands (PPB) was 99.1%. Shannon’s information index (I) and Nei’s gene diversity (He) from four populations were ordered (high to low) as herbstore > flourmill > bedroom > library. Population differentiation (Gst = 0.41) was average in these populations. Analysis of molecular variation (AMOVA) showed that variation existed mainly within populations (68.1%). At the same time, gene flow (Nm = 0.72) was interpreted to be moderate. Cluster analysis showed a small degree of genetic distance among different isolates and permitted grouping the isolates of L. bostrychophila into three distinct clusters. The study demonstrated clear genetic effects of habitat fragmentation on L. bostrychophila populations. Genetic diversity decreased and genetic differentiation increased between remnant populations when habitats were fragmented. Gene flow is not impeded by habitat fragmentation due to the impact of human activities. These findings are of great use for the prevention and control of this pest.     

Orientation of rusty grain beetles,Cryptolestes ferrugineus (Coleoptera: Laemophloeidae), to semiochemicals in field and laboratory experiments

Cryptolestes ferrugineus is a common beetle pest of stored grain for which techniques to monitor dispersing beetles are limited. Early research found several male-produced aggregation pheromones, but there has been little related work since that time. This paper reports experiments on orientation of C. ferrugineus in response to synthetic pheromones and other semiochemicals via flight and walking. Field trapping studies showed that flying beetles were caught on the western sides of grain bins in Kansas compared to the other three cardinal directions. Work with synthetic formulations of the two male-produced aggregation pheromones found that flying beetles were attracted to traps with the pheromone and wheat compared to traps with wheat only. Walking bioassays in the laboratory determined that either of the two aggregation pheromones, known as Cucujolide I and Cucujolide II, were attractive whether deployed singly or in combination. Laboratory bioassays showed that volatiles from grains, other grain-based materials and two commercial food attractants used in traps were attractive to C. ferrugineus. Further laboratory assays demonstrated that C. ferrugineus would orient to synthetic pheromones of three other beetle species and one moth species, all common pests of stored products. These new data on semiochemicals for C. ferrugineus suggest future work that could be done toward developing new tools for detecting and monitoring this serious pest.     

Monitoring flight activity of Cryptolestes ferrugineus (Coleoptera: Laemophloeidae) in outdoor environments using a commercial pheromone lure and the kairomone 1-octen-3-ol

Effective trapping techniques for Cryptolestes ferrugineus are essential for developing an understanding of the ecology of this beetle outside the storage environment, and will assist in monitoring the spread of fumigant resistance in field populations. We evaluated ten different volatile compounds associated with stored grains or fungi as potential attractants for C. ferrugineus using Lindgren funnel traps fitted with glass dispenser tubes. Only two compounds, 1-octen-3-ol and 3-octanone, were effective attractants in outdoor environments. Beetle captures increased with increasing release rates of both compounds. 1-octen-3-ol was the most effective attractant, and we evaluated it in conjunction with a commercial rubber septum lure containing the two macrocyclic lactones that constitute the C. ferrugineus aggregation pheromone. In parallel trials the commercial lure performed poorly, catching only 2% of the wild beetles recovered. 1-octen-3-ol deployed alone caught 26% of beetles, however a combination treatment (pheromone lure + 1-octen-3-ol) accounted for 72% of C. ferrugineus captures, suggesting a synergistic interaction. Evaluation of rubber septa impregnated with 1-octen-3-ol demonstrated that these were just as effective as glass release tubes when deployed with the pheromone lure over a five day period. A comparison of different trap types using the pheromone lure/1-octen-3-ol combined attractants showed that significantly more C. ferrugineus were captured with a panel trap than with either 4-unit or 9-unit Lindgren funnel traps, correlating to the much higher rate of 1-octen-3-ol volatilisation from the panel trap. A funnel trap fitted with a 1-octen-3-ol dispenser was used to continuously monitor C. ferrugineus flight activity for a three year period and captured 4335 individuals, demonstrating that although optimum catches are obtained using the pheromone/1-octen-3-ol combination, 1-octen-3-ol can be used alone to monitor the seasonal flight activity of C. ferrugineus.     

First report of strong phosphine resistance in stored grain insects in a far northern tropical region of Australia,combining conventional and genetic diagnostics

Although phosphine resistance monitoring and management programs are well advanced in Australia, the tropical northern part of the continent has received little attention. To address this, grain storages were sampled in the Townsville region in the Burdekin river catchment area of northern Queensland, and insect populations were subjected to resistance testing. Sampling of storages including silos, food processing facilities and feedlots was carried out during July to August 2019 and February to March 2020. Of the several species collected, populations of two major pest species, Rhyzopertha dominica and Tribolium castaneum were subjected to phenotypic resistance and molecular screening using discriminating doses and gene specific DNA marker, respectively. Utilising phenotypic assay, of the 17 populations of R. dominica, 12 were diagnosed as weakly resistant and five as strongly resistant; whereas of the 34 populations of T. castaneum, two were found to be susceptible to phosphine, 15 were determined as weakly resistant and 17 as strongly resistant. The molecular diagnostics, however, determined rph2 alleles for strong resistance in some populations each of R. dominica and T. castaneum that were diagnosed as weakly resistant in the phenotypic assay. The most prevalent rph2 alleles were found to be P49S and K142E in R. dominica populations and P45S and G131S in T. castaneum populations. Our results highlight the need of more comprehensive study towards determining the level of pests and resistance risks in this region.     

Development of microsatellite markers and a preliminary assessment of population structuring in the rice weevil,Sitophilus oryzae (L.)

The rice weevil, Sitophilus oryzae is one of the primary pests of stored grains worldwide. To develop and implement an effective integrated pest management strategy, an understanding of the population structuring of this destructive pest is vital. In this study we used Illumina paired-end sequencing to develop S. oryzae species-specific microsatellite markers, and used these markers to conduct a preliminary assessment of population structuring in four populations of S. oryzae from three countries (Australia, China, and USA). 7,635,996 raw sequencing reads were produced, with 11,794 microsatellites detected and 214,257 primer options designed. 48 microsatellite markers were selected for further validation, with 10 markers amplifying consistently across the four S. oryzae populations. These markers displayed a high level of polymorphism overall (6.67 alleles/locus), though this was slightly lower within populations (3.10–4.88 alleles/locus). We used the markers to conduct a preliminary assessment of genetic structuring among the four S. oryzae populations: three laboratory cultures (New South Wales, Queensland, and Santai) and a field collected population from Kansas. Analyses suggest high levels of genetic differentiation between the sample locations, with a global F_ST of 0.239, and pairwise F_ST values ranging from 0.100 to 0.395. Bayesian clustering analyses suggest these four populations formed four distinct clusters, with a similar pattern identified by Principal Coordinate Analysis. These microsatellite markers, together with our preliminary population genetic analyses, will provide a valuable resource for population genetic research, and contribute to effective integrated pest management strategies in the future.     

Protection of stored maize from insect pests using a two-component biological control method consisting of a hymenopteran parasitoid, Theocolax elegans, and transgenic avidin maize powder

The hymenopteran parasitoid, Theocolax elegans (Westwood), and transgenic avidin maize powder were tested to determine if their individual or combined use would protect stored grain from infestation by both internal and external insect pests. Small-scale tests were conducted in plastic jars containing 3 kg of non-transgenic maize. We tested treatments of 0.3% powdered avidin maize, the parasitoid wasp, and the combination of the parasitoid plus 0.3% powdered avidin maize. One pair each of Sitophilus zeamais Motschulsky, Tribolium castaneum (Herbst), and Cryptolestes ferrugineus (Stephens) was added to each jar. After 8 weeks, the entire contents of each jar were examined for adult insects. Control and avidin maize powders had no detrimental effects on the beneficial insect parasitoid T. elegans. The parasitoid suppressed populations of the internal feeder S. zeamais. The avidin maize powder treatment had no effect on S. zeamais because these larvae developed inside the maize kernels where no avidin maize powder was present. For S. zeamais, the combination treatment was not significantly different from the parasitoid treatment. In contrast, populations of the external feeder T. castaneum were not suppressed by the parasitoid but were suppressed by the avidin maize powder treatment. The parasitoid-avidin combination treatment produced the greatest percentage reduction for all three insect species and resulted in 78%, 94%, and 70% reductions in populations of S. zeamais, T. castaneum, and C. ferrugineus, respectively, when compared to the control treatment. The percentage reductions for the parasitoid treatment were 70%, 8%, and 20% for S. zeamais, T. castaneum, and C. ferrugineus, respectively. For the avidin maize powder treatment, populations of S. zeamais, T. castaneum, and C. ferrugineus were reduced by 10%, 85%, and 40%, respectively. The combination treatment of avidin maize powder plus the release of parasitoid wasps was superior to either treatment alone when applied to mixed populations of internal and external feeders.     

Molecular epidemiology and antimicrobial resistance profiles of Salmonella isolates from dairy heifer calves and adult lactating cows in a Mediterranean pasture-based system of Australia

Dairy cows can be reservoirs of foodborne pathogens such as Salmonella that pose serious public health risks to humans. The study was designed to examine the molecular epidemiology and antimicrobial resistance profiles of Salmonella isolates from dairy heifer calves and adult lactating cows in the pasture-based system of Australia. A total of 838 animals (328 heifer calves and 510 lactating cows) from 22 farms were sampled. Overall, 54 Salmonella isolates were recovered (calves 28/328 and cows 26/510). A herd-level Salmonella prevalence of 50% (95% confidence interval: 31%–69%) was recorded. Within-herd prevalence for Salmonella ranged between 4%–29% and 4%–45% among the heifer calves and adult lactating cows, respectively. Three different serovars were identified with Salmonella Infantis being the most common serovar (n = 33, 61%) followed by Salmonella Kiambu (n = 20, 37.0%) and one isolate of Salmonella Cerro (2%). The highest antimicrobial resistance prevalence of Salmonella isolates was found against streptomycin (n = 31, 57%), followed by cefoxitin (n = 12, 22%), ceftriaxone (n = 2, 4%), and chloramphenicol (n = 1, 2%). Multiple class resistance was observed on 4 isolates against cefoxitin, chloramphenicol, and streptomycin. Multilocus sequence types ST32 (61%), ST309 (37%), and ST367 (2%) were strongly linked to the serovars Salmonella Infantis, Salmonella Kiambu, and Salmonella Cerro, respectively. Whole genome sequencing of Salmonella isolates detected only 2 resistance genes: aac(6′) gene that confers resistance against aminoglycosides among 40.7% of the isolates, and a single isolate positive for the blaDHA-16 gene. Two distinct clusters among the serovars were observed suggesting 2 independent sources of spread. Despite the low prevalence of antimicrobial resistance among Salmonella from the dairy farms, our findings contribute to the regional and national understanding of antimicrobial resistance in dairy herds in Australia. There is need for continued antimicrobial resistance stewardship and surveillance programs to ensure the production of high-quality food products and the long-term protection of both animal and human health.     

转几丁质酶基因烟草遗传稳定性研究

从转几丁质酶基因烟草株系(NPTII基因是选择标记基因)对卡那霉素的抗性,及外源几丁质酶基因表达蛋白的活性(Western Blot检测)两个方面对转基因株系的遗传稳定性进行了分析研究。在卡那霉素抗性检测试验中,检测了13个T₂转基因株系,其中9个株系被检测种子100%对卡那霉素(100 mg/L)具有抗性;T₃和T₄转基因株系各检测了10个,100%被检测种子在含卡那霉素100 mg/L的MS培养基上长成了烟苗。Western Blot检测了11个T₂株系,检测结果表明,6个株系被检测植株100%含有外源几丁质酶蛋白,2个株系被检测植株80%含有外源几丁质酶蛋白,2个株系被检测植株50%含有外源几丁质酶蛋白,1个株系被检测植株19.05%含有外源几丁质酶蛋白。在温室内将转基因烟草与未转基因烟草密植在一起,种植2代,烟株开花期模仿自然风力对烟株吹风,进行了转基因烟草是否可以通过花粉进行基因漂移的研究。研究结果表明,在自然风媒条件下未发现转基因烟草可进行基因漂移。      

Effectiveness of a multi-species attractant in two different trap types under practical grain storage conditions

Commodities may be attacked by a multitude of pest species simultaneously and so to be cost-effective any lure must be attractive to a range of target species. The objective of this study was to test a multi-species lure formulation with different dispensers under conditions as close as possible to their use in practice. The attractant effect of the multi-species lure formulation was tested using two types of dispensers with populations of the three principal grain beetle pests in the UK (Oryzaephilus surinamensis, Sitophilus granarius and Cryptolestes ferrugineus). The trials were performed over a period of six weeks with the lures tested in PC™ Traps: first in a grain bulk and in six grain bins and second in the surrounding area using PC™ Floor Traps. The amounts of volatiles released in and around in situ traps were measured using Solid-Phase Microextraction (SPME).The first lure dispenser tested was attractive to O. surinamensis and C. ferrugineus but it attracted fewer S. granarius than the control traps. It was shown that most of the volatiles were released at the beginning of the trial and therefore lures were not effective over the whole six-week period. The second lure dispenser released the attractant volatiles more consistently over six weeks and exerted significant attraction to O. surinamensis and C. ferrugineus in PC™ Traps in the grain bulk and to O. surinamensis and S. granarius in floor traps. Too few C. ferrugineus were caught in floor traps to make a comparison. The reason for the repellent effect on S. granarius in the grain bulk was unclear and this is discussed along with the importance of measuring volatiles released by the lure.     

Effect of suboptimal temperatures and sublethal CO2 levels on multiplication of Tribolium castaneum (Coleoptera: Tenebrionidae), alone or competing with Cryptolestes ferrugineus (Coleoptera: Cucujidae)

Groups of adults of Tribolium castaneum alone, or in combination with adults of Cryptolestes ferrugineus, were exposed to suboptimal temperatures (15°C, 20°C, and 25°C) and sublethal CO₂ levels (2%, 5%, and 10%) in dry (12%, wet mass basis) and damp (15%, wet mass basis) stored wheat in the laboratory, to investigate effects on population size. The mean adult numbers in single- and mixed-species tests were positively correlated with higher temperature and moisture content and negatively correlated with higher CO₂ levels. Adult numbers in single- and mixed-species tests were lower at sublethal CO₂ levels compared to ambient CO₂ levels at all the test temperatures and decreased in dry grain compared to damp grain. Although, a specific trend was not observed in population inhibition between mixed-species and single-species tests, overall the adult populations of T. castaneum were reduced in the presence of C. ferrugineus. A mathematical model was derived to predict the size of adult populations of T. castaneum alone, or in the presence of C. ferrugineus considering all the variables in this study. The model had an R² value of 0.72 but needs to be validated and refined with field data.     

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.     

Movement of Cryptolestes ferrugineus (Coleoptera: Cucujidae) in grain columns containing pockets of high moisture content wheat and carbon dioxide gradients

The movement of the adult rusty grain beetle, Cryptolestes ferrugineus (Stephens), in 100 cm long, 10 cm diameter columns, filled with wheat of uniform or non-uniform moisture content (m.c.), was determined for horizontal and vertical placement of columns at 30°C.In horizontal columns filled with wheat of 13.5% uniform m.c., adult C. ferrugineus exhibited a trend of distributing themselves uniformly in the columns after 5 d. In the vertical columns filled with 14.7% uniform m.c. wheat, 44% of the insects moved downward after 7 d showing positive geotaxism. In the horizontal or vertical columns containing pockets of high m.c. wheat, 43-48% of the beetles aggregated in the region of high m.c. after 7 d exhibiting a hygrotactic response. Under CO₂ gradients in the columns filled with uniform m.c. wheat, 65% of C. ferrugineus moved towards higher levels of CO₂ after 1 and 5 d. Also, high levels of CO₂ were more influential in attracting C. ferrugineus (60%) after 1 d in horizontal columns containing high m.c. wheat at one end and high CO₂ levels at the other. In the vertical columns filled with pockets of high m.c. wheat and subjected to CO₂ gradients, movement of C. ferrugineus was more influenced by the combined effects of any two factors out of the three, viz., gravity, higher levels of CO₂ and high m.c. than a single factor.     

Characterization and horizontal transfer of class 1 integrons in Salmonella strains isolated from food products of animal origin

A total of 81 Salmonella isolates from retail meats and seafood in Hebei province, China, were assayed for the presence of and horizontal transfer of class 1 integrons. By the PCR screening for the integrons, class 1 integron was detected from strains in serotypes of Derby, Indiana, London and Choleraesuis, which were isolated from pork, chicken or seafood; however, two isolates contained the empty integron that lacked the resistance cassette, a potential hotspot for development of the multidrug resistance. In contrast, two other isolates had the antibiotic resistance gene cassettes within the class 1 integron, which were dfrA1-aadA1 and aadB-cmlA, respectively. The conjugation experiments demonstrated the plasmid-mediated transfer of the class 1 integrons. Furthermore, each of the integrons was transmitted to Streptococcus mutans via natural gene transformation. These findings suggest the possible transfer of class 1 integrons from foodborne pathogens to human residential bacteria via horizontal gene transfer.     

Genetic diversity, antimicrobial resistance and toxigenic profiles of Bacillus cereus isolated from food in Brazil over three decades

Bacillus cereus is an ever-present problem. It is widely distributed in several environments such as soil and plants and is commonly isolated from food and additives. In this study we analyzed 97 foodborne B. cereus sensu stricto strains isolated in Brazil in the 1980's, 1990's and 2000's in order to investigate the genetic diversity (assessed by Rep-PCR), antimicrobial resistance and toxigenic profiles (presence of hblA, hblC and hblD; nheA, nheB and nheC as well as cytK, ces and entFM genes) of such strains. The majority of the strains (79, 81.4%) were β-hemolytic. The NHE complex was found in 82 strains (84.5%) and HBL complex was found in 61 (62.9%) strains. All strains were negative to ces. The cytK-2 gene was found in 44 (45.4%) strains. The predominant toxigenic pattern was type I (32, 33%) which included strains positive for all toxin genes but ces. Computer assisted cluster analysis of Rep-PCR profiles showed a high genetic diversity. Seven major clusters comprising two or more strains were found and cluster 1 was predominant (ten strains, nine of them showing 100% similarity). This cluster included strains isolated in the 1980's and the 1990's. Cluster analysis of Rep-PCR profiles based on decade of isolation, source, hemolytic pattern, toxigenic and antibiotic resistance patterns revealed a similar clustering pattern as found in the analysis including all strains. The inability to observe a predominant band pattern when Rep-PCR cluster analysis was based on decade of isolation suggests that this diversity has been maintained over time. All strains were susceptible to gentamicin. We detected resistance to tetracycline (11 strains showing intermediate resistance and nine completely resistant strains), clindamycin (ten intermediate strains) and vancomycin (one strain). Clindamycin resistance showed statistical association with strains isolated in 2000's. The predominant resistance pattern was type A (72, 72.2%) which included strains susceptible to all drugs tested. Our results suggest that the majority of the strains present in several types of food in Brazil pose a potential risk to cause food poisoning due to the high prevalence of toxin genes found in these strains. However, additional studies involving cytotoxicity tests and affiliation of these strains to phylogenetic groups based on molecular data would be useful to better evaluate this potential and could provide a more accurate indication of the risk.     

A field demonstration of odour-based,host-food finding behaviour in several species of stored grain insects

In order to demonstrate odour-based, host-food finding behaviour in field populations of stored grain insects, traps were inserted into the fabric of PVC-covered, fully sealed, earth-walled storages at two sites in New South Wales, Australia. Cryptolestes ferrugineus, Rhyzopertha dominica and Tribolium castaneum were trapped relatively commonly, as were the economically less significant Carpophilus dimidiatus, Typhaea stercorea and Ahasverus advena. Trap odours from stored wheat contributed markedly to the attraction of all six species. Other species were taken also, mainly those associated with moist grain. These results are discussed in relation to the abundance of stored-product species in the trapping area.     

Numerical taxonomy of lactobacilli surviving radurization of meat

All 113 Gram-positive, catalase-negative, rod-shaped bacteria isolated from radurized (5 kGy) minced beef were homofermentative, aciduric and belonged to the sub-genus Streptobacterium. Lactobacillus sake was the predominant species with 100 strains being identified as such. Two strains produced L(+)-lactic acid and were identified as L. farciminis. Three strains were indistinguishable from L. curvatus and 8 strains were intermediate between L. sake and L. curvatus and were designated L. sake/curvatus. Numerical taxonomy by unweighted pair-group average linkage analysis revealed the existence of 5 clusters of these strains. Two isolates and all 7 reference strains were unclustered. Cluster 1 consisted of 4 sub-clusters (a-d) which all showed greater than 90% similarity. Clusters 2–5 were observed at 89, 88, 86 and 84% respectively. Cluster 1 contained 86 of the isolates which were split into sub-cluster: 1a (12 strains); 1b (44); 1c (17); 1d (13). All strains were closest to L. sake except for one strain in cluster 1b and five in cluster 1c which were L. sake/curvatus. Cluster 2 contained 9 isolates of which 3 strains were designated L. curvatus, one L. sake/curvatus and five L. sake. Cluster 3 contained 7 strains, 6 of which were L. sake and one was L. sake/curvatus. Cluster 4 contained 6 strains, all of which were L. sake and finally, cluster 5 contained 3 strains, two of which were L. sake whilst the other produced L(+)-lactic acid and was designated L. farciminis. DNA mol% G+C studies done on 7 L. sake isolates indicated a very wide range (37.3−44.2 mol%) of values within these strains.     

Mortality and movement of Cryptolestes ferrugineus and Rhyzopertha dominica in response to cooling in 300-kg grain bulks

We examined the effect of low temperatures on insect population density and mortality of adult and immature stages of Cryptolestes ferrugineus and Rhyzopertha dominica in three barrels holding 300 kg of wheat each. Barrels were infested with 2 insects/species/kg and left to develop at 30 °C for 5 months. Temperature of the grain mass before cooling was 38 °C in the centre to a low of 28 °C on the periphery. On 11 February the building door was opened to allow for cooling of the grain. During the 3 weeks of cooling, temperatures in the centre reached as low as −5 °C. Segregated (11 segments) grain trier samples were collected from three locations in each barrel, from February until March 2013 (6 dates), sieved, and the number of live and dead adults counted. Sieved grain was held for 5 weeks 30 °C and emerged adults counted to give an estimation of immature stages within the grain at the time of sampling. Rhyzopertha dominica adults were found mainly in the top 40 cm of the barrel, and there was little movement to the centre as the grain mass cooled. The initial distribution of C. ferrugineus adults was more complex; in two barrels adults were mostly in the centre of the grain mass, while in the other barrel, more adults were found on the periphery. When the grain mass cooled, C. ferrugineus adults in all barrels were found mostly in the centre. On day 0, the average number of live adults of C. ferrugineus and R. dominica were 232 ± 56 and 414 ± 48 insects/100 g, respectively. Rhyzopertha dominica adults were more susceptible to low temperatures than C. ferrugineus. The density of immatures of C. ferrugineus and R. dominica were similar at 0 d, there was a decline over the 21 d, but there was some survival at day 21.