Potential of the neonicotinoid imidacloprid and the oxadiazine indoxacarb for controlling five coleopteran pests of stored grain.

The potential for using imidacloprid (a neonicotinoid) and indoxacarb (an oxadiazine) as grain protectants was investigated in bioassays against resistant strains of five stored grain beetles. The species investigated were Rhyzopertha dominica (F.) (the lesser grain borer), Sitophilus oryzae (L.) (the rice weevil), Tribolium castaneum (Herbst) (the rust-red flour beetle), Oryzaephilus surinamensis (L.) (the saw tooth flour beetle), and Cryptolestes ferrugineus (Stephens) (the flat grain beetle). Each of these species has developed resistance to one or more protectants, including organophosphorus insecticides, synthetic pyrethroids and the juvenile hormone analogue methoprene. Mortality and reproduction after a 2-week exposure of adults to treated wheat depended on species, dose and insecticide. Imidacloprid had no effect on S. oryzae at any dose, but none of the other species produced any live progeny at 10 mg/kg. Indoxacarb had no effect on T. castaneum at any dose, but none of the other species produced any live progeny at 5 mg/kg. The results show that although both imidacloprid and indoxacarb can control at least four of the five key pests tested at doses comparable to those used for organophosphorus protectants, more potent neonicotinoid or oxadiazine insecticides would be needed than either of these to provide broad spectrum protection of stored grain.

Insect induced bud fall in cultivated hibiscus and aspects of the biology of Macroura concolor (Macleay) (Coleoptera: Nitidulidae)

The influence of insect attack on bud fall and subsequent poor flowering in cultivated hibiscus (Hibiscus rosa-sinensis) was studied in cages and in the field in southern Queensland. Three species of Hemiptera (most importantly Aulacosternum nigrorubrum but also Nezara viridula and Tectocoris diophthalmus) caused some bud fall in 2 plantations studied. Adults of Macroura concolor suppressed flowering for long periods in spring and summer. Data from white funnel traps and counts in flowers showed that M. concolor was most active in these seasons. Methiocarb (0.75 g a.i./litre) reduced beetle numbers and increased flowering. When 15 or more adults of M. concolor occurred per bud (or flower) most buds fell and few flowers were produced, but when beetles declined to 10 or fewer many buds survived and widespread flowering occurred. Larvae fed in fallen buds and flowers and the mean duration of development of the combined immature stages was 14 days at 26 deg C. The preference of adults of M. concolor for pale coloured flowers was examined. Hibiscus plants produced most buds from December to June with lower numbers in winter and spring (July to November). Bud production in spring and early summer (September-December) varied greatly and probably contributed to poor flowering, however, even when large numbers of buds occurred very few flowers were produced because of the activities of M. concolor.

Australian spore-feeding thrips of the genus Phaulothrips (Thysanoptera, Idolothripinae)

Breeding by several of the spore-feeding species in the genus Phaulothrips is shown to be associated with abandoned tunnels of bees and scolytid beetles, as well as with the dead seed capsules of Eucalyptus species. The breeding sites for other species in the genus remain unknown, but 16 species are here recognised from Australia, of which the following six are newly described: P. daguilaris, P. flindersi, P. kingae, P. kranzae, P. oakeyi, P. whyallae. Copyright © 2013 Magnolia Press.

Short-range resource location by Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) demonstrates a strong preference for fungi associated with cotton seed

Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) is a common stored grain pest for which a wide range of suitable resources has been recorded. These beetles are facultatively fungivorous and their resource range may extend to fungi associated with non-grain resources (e.g. cotton seed) and other decaying plant matter. Little is known with respect to fungi in terms of resource location by these beetles in the field. We, therefore, conducted a series of experiments in laboratory arenas, glasshouse cages and the field to determine how beetles respond to grain resources in relation to cotton seed (together with its lint stubble and associated fungal flora). Results from the tests conducted in relatively small arenas and cages in the laboratory and glasshouse reveal that the responses of T. castaneum adults to food resources were twice as strong when walking as when flying (as measured by the proportion of the released beetles that were trapped). Also, a clear preference for linted cotton seeds was evident in walking T. castaneum, especially in small-scale arenas in the laboratory, where at least 60% of beetles released preferred linted cotton seeds over wheat and sorghum. Similarly, in cages (1 m3) they responded five times more strongly to linted cotton seed than to conventional grain resources. However, this pattern was not consistent with those obtained from field trapping over 20 m and the beetles did not show any particular preference to any of the resources tested above. Our results suggest a focus on walking beetles in trapping studies for population estimations and, for developing effective food-based trapping lures, the potential use of active volatiles from the fungi associated with linted cotton seed. © 2012 Elsevier Ltd.

Responses of tribolium castaneum to olfactory cues from cotton seeds, the fungi associated with cotton seeds, and cereals

We tested, in an olfactometer, whether or not Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) responds preferentially to the volatiles that emanate from the fungi associated with cotton [Gossypium hirsutum L. (Malvaceae)] seed over those that emanate from cereals, because cereals are usually portrayed as the primary resources of these beetles. Pairwise comparisons were conducted between cotton seed, wheat (Triticum aestivum L.), and sorghum [Sorghum bicolor (L.) Moench] (both Poaceae); volatiles were tested from intact seeds and from both water and ethanol extracts. The results demonstrate that T. castaneum is attracted more strongly to cotton seeds with its lint contaminated with fungi, than to the conventional resources of this species (i.e., wheat and sorghum). Further tests prove that it is the fungus on the lint that produces the active volatiles, because the beetles did not respond to sterilized cotton lint (i.e., without the fungi typically associated with it when cotton seed is stored). Tests with five fungal cultures (each representing an unidentified species that was isolated from the field-collected cotton lint) were variable across the cultures, with only one of them being significantly attractive to the beetles. The others were not attractive and one may even have repulsed the beetles. The results are consistent with the beetles having a strong ecological association with fungi and suggest it would be worth investigating the ecology of T. castaneum from this perspective. © 2012 The Netherlands Entomological Society.

Determining changes in the distribution and abundance of a Rhyzopertha dominica phosphine resistance allele in farm grain storages using a DNA marker

BACKGROUND: The lesser grain borer, Rhyzopertha dominica (F.), is a highly destructive pest of stored grain that is strongly resistant to the fumigant phosphine (PH3). Phosphine resistance is due to genetic variants at the rph2 locus that alter the function of the dihydrolipoamide dehydrogenase (DLD) gene. This discovery now enables direct detection of resistance variants at the rph2 locus in field populations. RESULTS: A genotype assay was developed for direct detection of changes in distribution and frequency of a phosphine resistance allele in field populations of R. dominica. Beetles were collected from ten farms in south-east Queensland in 2006 and resampled in 2011. Resistance allele frequency increased in the period from 2006 to 2011 on organic farms with no history of phosphine use, implying that migration of phosphine-resistant R. dominica had occurred from nearby storages. CONCLUSION: Increasing resistance allele frequencies on organic farms suggest local movement of beetles and dispersal of insects from areas where phosphine has been used. This research also highlighted for the first time the utility of a genetic DNA marker in accurate and rapid determination of the distribution of phosphine-resistant insects in the grain value chain. Extending this research over larger landscapes would help in identifying resistance problems and enable timely pest management decisions. © 2013 Society of Chemical Industry © 2013 Society of Chemical Industry 69 6 June 2013 10.1002/ps.3514 Rapid Report Rapid Report © 2013 Society of Chemical Industry.

Constructing a new individual-based model of phosphine resistance in lesser grain borer (Rhyzopertha dominica): do we need to include two loci rather than one?

In this article, we describe and compare two individual-based models constructed to investigate how genetic factors influence the development of phosphine resistance in lesser grain borer (R. dominica). One model is based on the simplifying assumption that resistance is conferred by alleles at a single locus, while the other is based on the more realistic assumption that resistance is conferred by alleles at two separate loci. We simulated the population dynamic of R. dominica in the absence of phosphine fumigation, and under high and low dose phosphine treatments, and found important differences between the predictions of the two models in all three cases. In the absence of fumigation, starting from the same initial frequencies of genotypes, the two models tended to different stable frequencies, although both reached Hardy-Weinberg equilibrium. The one-locus model exaggerated the equilibrium proportion of strongly resistant beetles by 3.6 times, compared to the aggregated predictions of the two-locus model. Under a low dose treatment the one-locus model overestimated the proportion of strongly resistant individuals within the population and underestimated the total population numbers compared to the two-locus model. These results show the importance of basing resistance evolution models on realistic genetics and that using oversimplified one-locus models to develop pest control strategies runs the risk of not correctly identifying tactics to minimise the incidence of pest infestation.

Field Assessments of Control Agents for Lesser Mealworm (Coleoptera: Tenebrionidae) Using Litter Sampling

Spinosad, diatomaceous earth, and cyfluthrin were assessed on two broiler farms at Gleneagle and Gatton in southeastern Queensland, Australia in 2004-2005 and 2007-2009, respectively to determine their effectiveness in controlling lesser mealworm, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae). Insecticide treatments were applied mostly to earth or 'hard' cement floors of broiler houses before the placement of new bedding. Efficacy of each agent was assessed by regular sampling of litter and counting of immature stages and adult beetles, and comparing insect counts in treatments to counts in untreated houses. Generally, the lowest numbers of lesser mealworm were recorded in the house with hard floors, these numbers equalling the most effective spinosad applications. The most effective treatment was a strategic application of spinosad under feed supply lines on a hard floor. In compacted earth floor houses, mean numbers of lesser mealworms for two under-feed-line spinosad treatments (i.e., 2-m-wide application at 0.18 g of active insecticide (g [AI]) in 100-ml water/m(2), and 1-m-wide application at 0.11 g ([AI] in 33-ml water/m(2)), and an entire floor spinosad treatment (0.07 g [AI] in 86-ml water/m2) were significantly lower (i.e., better control) than those numbers for cyfluthrin, and no treatment (controls). The 1-m-wide under-feed-line treatment was the most cost-effective dose, providing similar control to the other two most effective spinosad treatments, but using less than half the active component per broiler house. No efficacy was demonstrated when spinosad was applied to the surface of bedding in relatively large volumes of water. All applications of diatomaceous earth, applied with and without spinosad, and cyfluthrin at the label rate of 0.02 g (AI)/100-ml water/m(2) showed no effect, with insect counts not significantly different to untreated controls. Overall, the results of this field assessment indicate that cyfluthrin (the Australian industry standard) and diatomaceous earth were ineffective on these two farms and that spinosad can be a viable alternative for broiler house use.

Response of Tribolium castaneum and Rhyzopertha dominica to various resources, near and far from grain storage

Tribolium castaneum (Herbst) and Rhyzopertha dominica (F.) are common cosmopolitan pests of stored grain and grain products. We evaluated the relative attraction of T.castaneum and R.dominica to wheat, sorghum and cotton seeds in the field, near grain storage facilities and well away from storages in southern and central Queensland using multiple trapping techniques. The results show that T.castaneum is more strongly attracted to linted cotton seed relative to wheat, whereas R.dominica did not respond to cotton seed at all and was attracted only to wheat. Significantly more adults of T.castaneum (10-15 times) were attracted to traps placed on the ground, near grain storage, than to equivalent traps that were suspended (1.5m above the ground) nearby. These results suggest that Tribolium beetles detect and respond to resources towards the end of their dispersal flight, after which they localize resources while walking. By contrast R.dominica was captured only in suspended traps, which suggests they fly directly onto resources as they localize them. The ability of both species to colonize and reproduce in isolated resource patches within the relatively short time of 1month is illustrated by the returns from the traps deployed in the field (at least 1km from the nearest stored grain) even though they caught only a few beetles. The results presented here provide novel insights about the resource location behaviours of both T.castaneum and R.dominica. In particular, the relationship of T.castaneum with non-cereal resources that are not conventionally associated with this species suggests an emphasis on these other resources in investigating the resource location behaviour of these beetles. This new perspective on the ecology of T. castaneum highlights the potential role of non-cereal resources (such as the lint on cotton seed) in the spread of grain pest infestations.

Rapid genome wide mapping of phosphine resistance loci by a simple regional averaging analysis in the red flour beetle, Tribolium castaneum

Background Next-generation sequencing technology is an important tool for the rapid, genome-wide identification of genetic variations. However, it is difficult to resolve the ‘signal’ of variations of interest and the ‘noise’ of stochastic sequencing and bioinformatic errors in the large datasets that are generated. We report a simple approach to identify regional linkage to a trait that requires only two pools of DNA to be sequenced from progeny of a defined genetic cross (i.e. bulk segregant analysis) at low coverage (<10×) and without parentage assignment of individual SNPs. The analysis relies on regional averaging of pooled SNP frequencies to rapidly scan polymorphisms across the genome for differential regional homozygosity, which is then displayed graphically. Results Progeny from defined genetic crosses of Tribolium castaneum (F4 and F19) segregating for the phosphine resistance trait were exposed to phosphine to select for the resistance trait while the remainders were left unexposed. Next generation sequencing was then carried out on the genomic DNA from each pool of selected and unselected insects from each generation. The reads were mapped against the annotated T. castaneum genome from NCBI (v3.0) and analysed for SNP variations. Since it is difficult to accurately call individual SNP frequencies when the depth of sequence coverage is low, variant frequencies were averaged across larger regions. Results from regional SNP frequency averaging identified two loci, tc_rph1 on chromosome 8 and tc_rph2 on chromosome 9, which together are responsible for high level resistance. Identification of the two loci was possible with only 5-7× average coverage of the genome per dataset. These loci were subsequently confirmed by direct SNP marker analysis and fine-scale mapping. Individually, homozygosity of tc_rph1 or tc_rph2 results in only weak resistance to phosphine (estimated at up to 1.5-2.5× and 3-5× respectively), whereas in combination they interact synergistically to provide a high-level resistance >200×. The tc_rph2 resistance allele resulted in a significant fitness cost relative to the wild type allele in unselected beetles over eighteen generations. Conclusion We have validated the technique of linkage mapping by low-coverage sequencing of progeny from a simple genetic cross. The approach relied on regional averaging of SNP frequencies and was used to successfully identify candidate gene loci for phosphine resistance in T. castaneum. This is a relatively simple and rapid approach to identifying genomic regions associated with traits in defined genetic crosses that does not require any specialised statistical analysis.

Host Tree Influences on Longicorn Beetle (Coleoptera: Cerambycidae) Attack in Subtropical Corymbia (Myrtales: Myrtaceae)

Phoracantha longicorn beetles are endemic to Australia, and some species have become significant pests of eucalypts worldwide, yet little is known about their host plant interactions and factors influencing tree susceptibility in Australia. Here, we investigate the host relationships of Phoracantha solida (Blackburn, 1894) on four eucalypt taxa (one pure species and three hybrid families), examining feeding site physical characteristics including phloem thickness, density, and moisture content, and host tree factors such as diameter, height, growth, taper, and survival. We also determine the cardinal and vertical (within-tree) and horizontal (between-tree) spatial distribution of borers. Fewer than 10% of P. solida attacks were recorded from the pure species (Corymbia citriodora subsp. variegate (Hook)), and this taxon also showed the highest survival, phloem thickness, relative growth rate, and bark:wood area. For the two most susceptible taxa, borer severity was negatively correlated with moisture content, and positively related to phloem density. Borers were nonrandomly and nonuniformly distributed within trees, and were statistically aggregated in 32% of plots. More attacks were situated on the northern side of the tree than the other aspects, and most larvae fed within the lower 50 cm of the bole, with attack height positively correlated with severity. Trees with borers had more dead neighbors, and more bored neighbors, than trees without borers, while within plots, borer incidence and severity were positively correlated. Because the more susceptible taxa overlapped with less susceptible taxa for several physical tree factors, the role of primary and secondary chemistries in determining host suitability needs to be investigated. Nevertheless, taxon, moisture content, phloem density, tree size, and mortality of neighboring trees appeared the most important physical characteristics influencing host suitability for P. solida at this site.

Aiming for the management of the small hive beetle, Aethina tumida, using relative humidity and diatomaceous earth

Small hive beetles (SHBs) are a global pest of European honeybee colonies. In the laboratory, the survival of adult SHBs was evaluated in relation to relative humidity (RH = 56, 64, 73, 82 and 96 %) and treatment with diatomaceous earth (DE) across 4 days. Low RH reduced survival. The application of DE reduced survival in addition to RH. Adults treated with corn flour (control) showed no difference in survival from untreated beetles. Scanning electron microscopy images showed no scarification of adult beetle cuticle after exposure to DE; therefore, water loss is likely facilitated through non-abrasive means such as the adsorption of cuticular lipids. The data agree with the hypothesis that DE causes mortality through water loss from treated insects. Egress, ingress, mortality and the egg-laying behaviours of beetles were observed in relation to a popular in-hive trench trap with and without the addition of DE. Traps filled with DE resulted in 100 % mortality of beetles compared with 8.6 % mortality when no DE was present. A simple method for visually determining beetle sex was used and documented.

Dietary media for mass rearing of rusty grain beetle, Cryptolestes ferrugineus (Stephens) and flat grain beetle, Cryptolestes pusillus (Schonherr) (Coleoptera: Cucujidae)

We developed a suitable diet for mass rearing of Cryptolestes ferrugineus (Stephens) populations under laboratory conditions. Recently, this pest has developed strong level of resistance to phosphine in Australia, and therefore, a significant amount of research has been directed towards its management. In total, nineteen grain-based diets, containing rolled oats, various combinations of cracked grains and flours of wheat, sorghum, maize and barley were tested. Each diet contained a small proportion of wheat germ (4.5% w/w) and torula yeast (0.5% w/w). Experiments were conducted at fixed temperature and relative humidity regimes of 30 ± 2 °C and 70 ± 2%, respectively, and replicated three times. Adults (n = 40) of a laboratory strain of C. ferrugineus were introduced into each diet, removed after 14 days and total numbers of live adult progeny were recorded. The following diets resulted in highest live progeny production: barley flour (95%) (607.67 ± 11.21) = rolled oats (75%) + cracked sorghum (20%) (597.33 ± 33.79) ≥ wheat flour (47.5%) + barley flour (47.5%) (496.67 ± 52.93) > cracked sorghum (95%) (384.00 ± 60.66). The performance of these four diets was then tested with field-collected populations of C. ferrugineus and Cryptolestes pusillus (Schonherr). The diets based on rolled oats + cracked sorghum, wheat flour + barley flour, and barley flour alone consistently produced highest progeny numbers in field-collected populations of both species, with mean progeny numbers ranging from 359.9 to 478.5. The multiplication of C. pusillus was significantly higher than C. ferrugineus on all four diets. Our findings will help in mass rearing of healthy cultures of C. ferrugineus and C. pusillus that will greatly facilitate laboratory and field research and in particular, in developing management tactics for these species.

Do Phosphine Resistance Genes Influence Movement and Dispersal Under Starvation?

Phosphine resistance alleles might be expected to negatively affect energy demanding activities such as walking and flying, because of the inverse relationship between phosphine resistance and respiration. We used an activity monitoring system to quantify walking of Rhyzopertha dominica (F.) and a flight chamber to estimate their propensity for flight initiation. No significant difference in the duration of walking was observed between the strongly resistant, weakly resistant, and susceptible strains of R. dominica we tested, and females walked significantly more than males regardless of genotype. The walking activity monitor revealed no pattern of movement across the day and no particular time of peak activity despite reports of peak activity of R. dominica and Tribolium castaneum (Herbst) under field conditions during dawn and dusk. Flight initiation was significantly higher for all strains at 28 degrees C and 55% relative humidity than at 25, 30, 32, and 35 degrees C in the first 24 h of placing beetles in the flight chamber. Food deprivation and genotype had no significant effect on flight initiation. Our results suggest that known resistance alleles in R. dominica do not affect insect mobility and should therefore not inhibit the dispersal of resistant insects in the field.

Diagnostic Molecular Markers for Phosphine Resistance in U.S. Populations of Tribolium castaneum and Rhyzopertha dominica

Stored product beetles that are resistant to the fumigant pesticide phosphine (hydrogen phosphide) gas have been reported for more than 40 years in many places worldwide. Traditionally, determination of phosphine resistance in stored product beetles is based on a discriminating dose bioassay that can take up to two weeks to evaluate. We developed a diagnostic cleaved amplified polymorphic sequence method, CAPS, to detect individuals with alleles for strong resistance to phosphine in populations of the red flour beetle, Tribolium castaneum, and the lesser grain borer, Rhyzopertha dominica, according to a single nucleotide mutation in the dihydrolipoamide dehydrogenase (DLD) gene. We initially isolated and sequenced the DLD genes from susceptible and strongly resistant populations of both species. The corresponding amino acid sequences were then deduced. A single amino acid mutation in DLD in populations of T.castaneum and R.dominica with strong resistance was identified as P45S in T.castaneum and P49S in R.dominica, both collected from northern Oklahoma, USA. PCR products containing these mutations were digested by the restriction enzymes MboI and BstNI, which revealed presence or absence, respectively of the resistant (R) allele and allowed inference of genotypes with that allele. Seven populations of T.castaneum from Kansas were subjected to discriminating dose bioassays for the weak and strong resistance phenotypes. Application of CAPS to these seven populations confirmed the R allele was in high frequency in the strongly resistant populations, and was absent or at a lower frequency in populations with weak resistance, which suggests that these populations with a low frequency of the R allele have the potential for selection of the strong resistance phenotype. CAPS markers for strong phosphine resistance will help to detect and confirm resistant beetles and can facilitate resistance management actions against a given pest population.