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.     

Use of macro and trace elements as biological markers in the lesser grain borer,Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae)

The relationship between the occurrence and movements of Rhyzopertha dominica (F.), a serious pest of stored cereal grains, in distinctly different habitats is poorly known. Understanding the feeding ecology and dispersal patterns of R. dominica may likely help predict the abundance or movement of this pest in various habitats. Natal sites and movement of insects are studied using intrinsic methods involving elemental analyses. In this study, to understand the dietary history of R. dominica, trace and macro elements were used as potential markers. Insects reared on selected diets under laboratory conditions and adult beetles collected from the periphery of granaries or inside forested woodlands were used to determine 10 different elements in their body tissues. Quantities of 10 elements were also determined in respective laboratory hosts. The main discriminant elements for laboratory-reared R. dominica were Na, S and Zn, while significantly different elements were distinctive among field-collected beetles and included P, Ca, K, Zn, and Cu. The amount of Zn quantified in acorn-fed lab reared beetles was significantly lower than wheat- or corn-fed insects. Similarly, beetles captured in woodlands had significantly lower Zn in their body signatures, suggesting acorn seeds may have served as an alternate host in Quercus spp. dominated woodlands. Our studies showed that elemental profiles may potentially be used as biological markers for delineating food sources of R. dominica to mainly discriminate whether beetles originated in agricultural vs. wooded habitats.     

Significant population structure in Australian Cryptolestes ferrugineus and interpreting the potential spread of phosphine resistance

An analysis of the population genetics of Cryptolestes ferrugineus, across all major regions in Australia where grain is grown and stored in bulk, provides an initial step in investigating the movement of these insects and implications for the spread of phosphine resistance. Microsatellite data revealed two levels of genetic structure. Across Australia, two clusters were detected, one in eastern Australia (Queensland, New South Wales, Victoria and Tasmania) and Western Australia (Cluster 1) and one in South Australia (Cluster 2). Intermediate between them, in eastern South Australia, are a couple of populations that are equally admixed for the two clusters. Populations in Western Australia and western SA belong to different clusters suggesting that the extensive and arid Nullarbor Plain restricts the natural dispersal of these beetles. Isolation-by-distance and the detection of clustering among local populations suggests there is considerable gene flow at a local scale in C. ferrugineus, and we infer this is by flight. Limited natural gene flow between eastern and western Australia might, in part, explain why extremely high resistance has not been detected in Western Australia.     

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.     

Proteomic assessment of resistance to the fumigant phosphine in the lesser grain borer, Rhyzopertha dominica (F.)

A previous publication compared a strain of the stored-grain pest beetle, Rhyzopertha dominica, resistant to the fumigant phosphine, with a susceptible strain using two-dimensional polyacrylamide gel electrophoresis. Certain proteins appeared to differ and the authors proposed that arginine kinase in particular might be used as a marker for the rapid monitoring of resistance in this species and other pests of stored grain. Here an expanded set of susceptible and resistant strains is surveyed using the two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) system. Of hundreds of spots only two showed a significant difference between the resistant and susceptible strains and the magnitudes of those differences were less than three-fold. Arginine kinase spots were identified but they did not differ significantly. The proposal that certain abundant proteins of R. dominica might be used as markers of phosphine resistance was not supported by the present study.     

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.     

Testing the consistency of spatio-temporal patterns of flight activity in the stored grain beetles Tribolium castaneum (Herbst) and Rhyzopertha dominica (F.)

Many studies have been published on the flight activity of stored grain beetles in temperate regions, but few have focussed on tropical and sub-tropical regions. The spatio-temporal patterns of flight activity of Tribolium castaneum (Herbst) and Rhyzopertha dominica (F.) were quantified in a grain growing district on the Tropic of Capricorn in central Queensland, Australia. Nine traps baited with aggregation pheromone lures were monitored at 2-wk intervals (fortnightly) for 1 year along a 28.4 km linear transect that included sites at bulk grain depots and sites away from stored grain. Beetles of both species were trapped every fortnight during the study. The spatio-temporal patterns of flight activity differed greatly across the two species, as predicted from studies elsewhere. Rhyzopertha dominica was widespread across the landscape, as the mean trap catch of this species was equal in depot and non-depot traps. In contrast, T. castaneum was more frequently trapped in depot traps than non-depot traps during the colder months, but was much more widespread across the landscape during the summer months. Tribolium castaneum also showed a clear mid-summer peak in flight activity, whereas R. dominica flight activity was highly variable throughout the study. In general, our results reveal patterns that are consistent with those found for T. castaneum and R. dominica in southern Queensland. The contrasting spatio-temporal patterns of flight activity of R. dominica and T. castaneum show that species-specific approaches may be needed to manage these pests and that the spatio-temporal dynamics of resistance genes may differ across these species.     

Mobility parameters of Tribolium castaneum and Rhyzopertha dominica populations with different susceptibility to phosphine

Phosphine (PH₃) is the most commonly used fumigant to protect stored products from arthropod infestations worldwide. Our knowledge about the behavioral differences between phosphine-resistant and -susceptible stored product pest populations is limited. This study evaluated differences in mobility and behavior of populations of two major stored product insects, Tribolium castaneum (Herbst) and Rhyzopertha dominica (F.), which have different susceptibility to phosphine. In this regard, laboratory bioassays in Petri dish arenas were designed to determine if phosphine resistance has an impact on the walking and mobility behavior of adult beetles of both species. Results indicated that there were significant differences between resistant and susceptible populations for both species. Regarding velocity, R. dominica susceptible individuals moved faster than resistant ones. However, the resistant population showed reduced activity for several parameters tested compared to the susceptible population. Similar trends were also noted for T. castaneum. Knowledge of these parameters should be further utilized in management tactics, as resistant populations may behave in a different way in key management indicators such as trapping and sampling, as compared with susceptible ones.     

Microsatellite markers for Rhyzopertha dominica (Coleoptera: Bostrichidae)

Rhyzopertha dominica (F.) is a destructive insect pest of stored products. Due to this pest's broad host range, short life cycle, and high reproductive capacity, it has rapidly spread throughout the world. Understanding the population structuring of this important pest is vital for developing and implementing an effective integrated pest management strategy. In this study, we isolated 19 novel polymorphic microsatellite loci from an enriched genomic library based on a biotin/streptavidin capture protocol. The genetic parameters were estimated based on 80 individual R. dominica from two natural populations. The numbers of alleles ranged from 3 to 10, and sixteen loci exhibited polymorphic information contents (PICs) greater than 0.5. The observed (H_O) and expected (H_E) heterozygosities ranged from 0.033 to 0.900 and 0.310 to 0.882, respectively. Six locus/population combinations significantly deviated from Hardy–Weinberg equilibrium (HWE). These microsatellite markers will provide a valuable resource for studying the population genetic structure of this species.     

Toxicity of coumaran to stored products beetles

The fumigant activity of coumaran was tested against mixed-age cultures of the rice weevil (Sitophilus oryzae L.), the lesser grain borer (Rhyzopertha dominica F.), the rust-red flour beetle (Tribolium castaneum Herbst), saw-toothed grain beetle (Oryzaephilus surinamensis L.) and flat grain beetle (Cryptolestes ferrugineus Stephens). T. castaneum was tolerant to coumaran, for total population extinction was achieved in 72 h exposure at 50 μg/L dose only. In contrast all developmental stages of O. surinamensis, C. ferrugineus and R. dominica were quite susceptible 100% mortality was attained at < 50 μg/L doses in 24 or 72 h exposure. Further results showed that T. castaneum, generally tolerant pest to fumigants and R. dominica, a predominant stored grain pest in India, could be controlled by coumaran. The findings indicate that, coumaran is good source for alternate to methylbromide for effective control of stored grain insect pests.     

Effectiveness of spinosad as a grain protectant against resistant beetle and psocid pests of stored grain in Australia

Effectiveness of the bacterium-derived insecticide, spinosad, was determined against eight storage pests of Australia. Laboratory experiments were carried out on relevant resistant strains of four beetle and four psocid species, with the aim of determining the potential of spinosad as a new grain protectant. To explore the possibility that spinosad could have delayed effects, we exposed all insects for 14 d initially and then a further 14 and 28 d for psocids and beetles, respectively. Adult insects of each strain were exposed to untreated wheat (control) and wheat treated with spinosad at 0.1, 0.5 and 1 mg [a.i.]/kg of grain, and adult mortality and reduction of progeny were determined. Among beetles, spinosad was most effective against Rhyzopertha dominica (F.), with 100% adult mortality and progeny reduction after 14 d exposure at 1 mg [a.i.]/kg. Efficacy of spinosad was less with Sitophilus oryzae (L.), and least with Tribolium castaneum (Herbst) and Oryzaephilus surinamensis (L.). Against the psocids, spinosad was most effective against Liposcelis entomophila (Enderlein), with 100% adult mortality after 28 d exposure at 1 mg [a.i.]/kg and 92% progeny reduction after 14 d exposure and 100% subsequently. Spinosad was only moderately effective against Liposcelis bostrychophila Badonnel, L. decolor (Pearman) and L. paeta Pearman. Our findings suggest spinosad to be a potential protectant against R. dominica and L. entomophila in stored grain in Australia. This potential use would be in combination with another protectant capable of controlling other members of the pest complex.     

Aggregation pheromone 4,8-dimethyldecanal and kairomone affect the orientation of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) adults

Tribolium castaneum, the red flour beetle, is a major pest of stored products throughout the world. The male T. castaneum adults biosynthesize and release the aggregation pheromone 4,8-dimethyldecanal (4,8 DMD), and the monitoring traps having the synthetic form of this pheromone and kairomone are commercially available. However, certain information available on the response of T. castaneum adults to the strength of pheromone and the nature of air flow is inadequate and thus limits the potential use of this compound. The objectives of this research were to determine if the orientation of adult beetles to the synthetic form of the pheromone 4,8 DMD is affected by the distance from the pheromone, its concentration and air movement. Experimental design was completely randomized design with three replicates. The T. castaneum adults released at different distances from the pheromone placed at varying concentrations tested their orientation to the pheromone with the presence or absence of air flow. The control experiments were conducted without the pheromone. A separate experiment tested if exposure to 4,8 DMD alters the direction of movement in T. castaneum adults. The maximum trap catch (24%) was obtained with 0.5 μL of 4,8 DMD and when the beetles were released at distances up to 60 cm from the pheromone. The percentage of beetles trapped declined when the beetles were released 60–120 cm from the pheromone. The average angles on beetle turning during their orientation were significantly higher in the presence of pheromone than the respective controls. Air movement downstream of the pheromone trap increased the percentage beetles trapped compared to that in the absence of air flow. It is concluded that the orientation of T. castaneum adults is affected by the concentration of 4,8 DMD, distance from the pheromone and the air movement.     

Do walking Rhyzopertha dominica (F.) locate cereal hosts by chance?

To clarify the role of host odours in host location by walking Rhyzopertha dominica, locomotory responses of beetles were investigated in an experimental arena. Movements of beetle downwind of clean wheat, brown rice or maize, or wheat infested by conspecifics, were recorded by video and analysed using motion analysis software. No differences could be detected in the responses of beetles to the odours of clean wheat or to clean air; they showed equally strong positive anemotaxis to both. On average, only 37% of beetles arrived at the three clean food sources tested while 80% were able to locate the infested wheat. Both sexes showed an orientation component (taxis) in their behaviour towards the infested food source; females turned more (positive klinokinesis) and walked faster (positive orthokinesis) while there was no difference in male velocity between the odours of clean or infested wheat. There was no evidence of response to host odours and the locomotory responses of beetles that were able to locate clean wheat were similar to those that failed to do so. Investigation of beetles in potentially different physiological states, i.e. those that had actively dispersed from a food source or females reared in an environment isolated from other insects, showed no behavioural responses to wheat volatiles. Together, these studies gave no evidence that walking R. dominica use host volatiles to locate cereals. On the contrary, initial host location of cereals may occur by chance, as would seem to be the case in the closely related Prostephanus truncatus. The implications of this for better pest management of R. dominica are discussed.     

Geostatistical analysis of adult Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) in wheat stored at constant temperatures

Insect monitoring and sampling programmes are used in the stored grains industry for the detection and estimation of insect pests. At the low pest densities dictated by economic and commercial requirements, the accuracy of both detection and abundance estimates can be influenced by variations in the spatial structure of pest populations over short distances. Geostatistical analysis of Rhyzopertha dominica populations in 2-dimensions showed that, in both the horizontal and vertical directions and at all temperatures examined, insect numbers were positively correlated over short (0–5 cm) distances, and negatively correlated over longer (≥10 cm) distances. Analysis in 3 dimensions showed a similar pattern, with positive correlations over short distances and negative correlations at longer distances. At 35 °C, insects were located significantly further from the grain surface than at 25 and 30 °C. Dispersion metrics showed statistically significant aggregation in all cases. This is the first research using small sample units, high sampling intensities, and a range of temperatures, to show spatial structuring of R. dominica populations over short distances. This research will have significant implications for sampling in the stored grains industry.     

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.     

Comparative performance of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) across populations,resource types and structural forms of those resources

The significance of Tribolium castaneum (Herbst) as a pest of stored grains will likely be influenced by the relative availability of different resources regionally and the beetles’ ability to utilize each of them. Therefore the performance across the life cycle of laboratory and field populations of T. castaneum on commonly available structural forms of wheat and sorghum, as conventional resources, was quantified and compared to that on cotton seed. The latter is not usually associated with T. castaneum, but large beetle infestations have been recorded commonly in cotton gins in Australia. The highest number of individuals of each stage (from a given number of eggs) developed on wheat and sorghum flour and kibbled seeds. Cotton seed did not support the development of laboratory population beetles and all neonate larvae died before pupation. In contrast, some of the field population larvae did develop on cotton flour, but their number was significantly lower in comparison to development on wheat and sorghum. Field population beetles produced significantly more eggs than did laboratory population beetles, on both the whole seed and flour. Field population females had a consistently higher fecundity (～78% more eggs) than the laboratory population females, at least over the first three weeks of adult life. These results suggest that field populations of T. castaneum should be used in experiments if we are to develop a more realistic understanding of the field ecology of this species. The relative rate at which these insects infest available resources in the field also warrants further investigation.     

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.     

Evaluation of dosimeter tubes for monitoring phosphine fumigations

Within integrated pest management options, fumigation of stored products is one method to help control post-harvest insect infestations in our food and agricultural products. Fumigant gas concentration monitoring is important to confirm that the treatment was adequate to achieve the desired insect control, but monitoring can be relatively expensive and labor intensive. This study evaluated how accurately dosimeter tubes could monitor phosphine fumigation treatments. The dosimeter tube is designed to continuously react with phosphine gas during the fumigation period and yields a measurement in terms of concentration 1 time product or CT, which can be interpreted as cumulative exposure. Two models of dosimeter tubes were evaluated (high range and low range). The reference method for these trials were wireless phosphine monitoring sensors, which recorded gas concentrations at hourly intervals during an exposure, and from this a CT product was also calculated. Model LPG-1, high-range dosimeter tube, measured within ± 25% of the phosphine monitoring sensors for CT dosages less the 70,000 ppm1hr. Model LPG-2, low-range tube, tended to significantly over-estimate phosphine CT dosage by 50%–100% of the phosphine monitoring sensor references. Secondly, bioassays of fumigant efficacy were performed using susceptible and resistant adult Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), lesser grain borers, and Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), red flour beetle, for estimating insect control at the varied fumigation CT treatments. For the susceptible strains, CT dosages ∼5000 ppm1hr controlled both species. However, the insect control varied from 60% to 100% for resistant adults at CT dosages of ∼20,000 ppm1hr. The dosimeter tubes function in these ranges of dosages where each insect species are controlled and the dosimeter tube model LPG-1 provides reasonable estimates of the fumigation dosage for a given treatment level.     

A review of the biology and control of Rhyzopertha dominica (F.) the lesser grain borer

The lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae) is a primary pest of stored grains in many regions of the world. A literature review was done to present a summary of relevant literature on the general biology and ecology of the insect. The review addresses the following general subjects: first, a taxonomic discussion, which includes a review of other stored product bostrichid insects; second, details on the courtship behavior and conditions favorable for pest development; third, a discussion of the life stages; fourth, a review of R. dominica feeding habits, which includes a review of various grain and non-grain substances that have been reported as forming food and potential breeding resources for the insect, and discussion on grain losses due to R. dominica infestation; fifth, a discussion on the microorganisms and natural enemies associated with the insect; sixth, a summary of its flight behavior and flight activity, which includes a review of factors influencing flight initiation, daily and seasonal flight activity; seventh, a review of host location mechanism; eight, details on the biology of its aggregation pheromones; and ninth, a discussion on control; and lastly, a summary of the review. The review also identifies potential areas of further research on R. dominica that include, but are not limited to the role of arboreal food and forest floral in the population dynamics and life history of the insect, investigation of the mechanism by which the insect orients to host material not associated with pheromones, and studies on the site and mechanism of pheromone biosynthesis in the insect.