New and rapid visual detection assay for Trogoderma granarium everts based on recombinase polymerase amplification and CRISPR/Cas12a.

BACKGROUND: Khapra beetle (Trogoderma granarium Everts), one of the most important quarantine pests globally, is capable of causing severe infestation and huge economic loss to stored grain, and its interception rate has increased in major global trade countries over the past few years. However, difficulties remain in distinguishing this species with similar ones. In order to assist border ports and warehouses in khapra beetle's effective rapid identification as well as pest control at the early stages of monitoring or interception, we herein developed a new and rapid visual detection assay for T. granarium based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system. RESULTS: We designed and selected the first khapra beetle-specific RPA primers and crRNA, and optimized the visualization reaction system (Cas12a/CrRNA = 100 nM/500 nM). With only a 37 °C-heat-source and a blue light torch, RPA and CRISPR/CAS12a-based visualization assays can be completed within 40 min to differentiate between khapra beetle and nine similar Dermestidae species. After DNA extraction using a kit (4-5 h) or a simple method (5 min), the specific amplicons were obtained after a 15 min RPA reaction at 37 °C, followed by a 15 min color reaction under 37 °C in dark conditions using a CRISPR/CAS12a system and a fluorescent probe (5'-FAM/3'-BHQ1 labeled). This method is ingenious to low levels of DNA (10-1 ng µL-1 ) and meets the sensitivity requirements for detecting a single khapra beetle's egg (≈0.7 mm). CONCLUSION: Our specificity and sensitivity analysis inferred that the present visualization system is effective to quickly and uniquely detect khapra beetle at room temperature (37 °C), thereby preventing this species before they spread widely. Our study is suitable for being pushed forward in storage pest management, and provides value as a reference for monitoring and identification of other pests. © 2023 Society of Chemical Industry.

Evaluation of Phosphine Resistance in Populations of Sitophilus oryzae, Oryzaephilus surinamensis and Rhyzopertha dominica in the Czech Republic.

Phosphine is globally the most widely adopted fumigant for the control of storage pests. Recently, an increase in the frequency of stored-product pest resistance has been observed with significant geographical and interspecific variations. In this context, there are available data for the occurrence of resistant populations from America, Asia, Africa, and Australia, but there are few data in the case of Europe. Therefore, the aim of this work was to evaluate phosphine efficacy in important beetle pests of stored products, i.e., Sitophilus oryzae (L.), Oryzaephilus surinamensis (L.), and Rhyzopertha dominica (F.) sampled from the Czech Republic, using a rapid diagnostic test that is based on the speed to knockdown after exposure. Apart from the standard laboratory populations, which were used as the controls, we tested 56 field populations of these three species, collected in Czech farm grain stores. The survey revealed that 57.1% of the tested field populations were classified as phosphine-susceptible, based on the knockdown method used. However, profound variations among species and populations were recorded. The species with the highest percentage of resistant populations was R. dominica (71.4% of the populations; resistance coefficient 0.5-4.1), followed by S. oryzae (57.1% of the populations; resistance coefficient 0.8-6.9), and O. surinamensis (9.5% of the populations; resistance coefficient 0.5-2.9). Regarding the intra-population variability in response to phosphine (slope of the knockdown time regression), the laboratory and slightly resistant populations of all species were homogenous, whereas the most resistant populations were strongly heterogeneous. Our data show that the occurrence of resistance in the Czech Republic is relatively widespread and covers a wide range of species, necessitating the need for the adoption of an action plan for resistance mitigation.

Gel Carriers for Plant Extracts and Synthetic Pesticides in Rodent and Arthropod Pest Control: An Overview.

Insecticides and rodenticides form the basis of integrated pest management systems worldwide. As pest resistance continues to increase and entire groups of chemical active ingredients are restricted or banned, manufacturers are looking for new options for more effective formulations and safer application methods for the remaining pesticide ingredients. In addition to new technological adaptations of mainstream formulations in the form of sprays, fumigants, and dusts, the use of gel formulations is becoming increasingly explored and employed. This article summarizes information on the current and potential use of gel (including hydrogel) and paste formulations against harmful arthropods or rodents in specific branches of pest management in the agricultural, food, stored product, structural wood, urban, medical, and public health areas. Due to the worldwide high interest in natural substances, part of the review was devoted to the use of gels for the formulation of pesticide substances of botanical origin, such as essential or edible oils. Gels as emerging formulation of so called "smart insecticides" based on molecular iRNA disruptors are discussed.

Synthetic and Natural Insecticides: Gas, Liquid, Gel and Solid Formulations for Stored-Product and Food-Industry Pest Control.

The selective application of insecticides is one of the cornerstones of integrated pest management (IPM) and management strategies for pest resistance to insecticides. The present work provides a comprehensive overview of the traditional and new methods for the application of gas, liquid, gel, and solid physical insecticide formulations to control stored-product and food industry urban pests from the taxa Acarina, Blattodea, Coleoptera, Diptera, Hymenoptera, Lepidoptera, Psocoptera, and Zygentoma. Various definitions and concepts historically and currently used for various pesticide application formulations and methods are also described. This review demonstrates that new technological advances have sparked renewed research interest in the optimization of conventional methods such as insecticide aerosols, sprays, fumigants, and inert gases. Insect growth regulators/disruptors (IGRs/IGDs) are increasingly employed in baits, aerosols, residual treatments, and as spray-residual protectants for long-term stored-grain protection. Insecticide-impregnated hypoxic multilayer bags have been proven to be one of the most promising low-cost and safe methods for hermetic grain storage in developing countries. Insecticide-impregnated netting and food baits were originally developed for the control of urban/medical pests and have been recognized as an innovative technology for the protection of stored commodities. New biodegradable acaricide gel coatings and nets have been suggested for the protection of ham meat. Tablets and satchels represent a new approach for the application of botanicals. Many emerging technologies can be found in the form of impregnated protective packaging (insect growth regulators/disruptors (IGRs/IGDs), natural repellents), pheromone-based attracticides, electrostatic dust or sprays, nanoparticles, edible artificial sweeteners, hydrogels, inert baits with synthetic attractants, biodegradable encapsulations of active ingredients, and cyanogenic protective grain coatings. Smart pest control technologies based on RNA-based gene silencing compounds incorporated into food baits stand at the forefront of current strategic research. Inert gases and dust (diatomaceous earth) are positive examples of alternatives to synthetic pesticide products, for which methods of application and their integration with other methods have been proposed and implemented in practice. Although many promising laboratory studies have been conducted on the biological activity of natural botanical insecticides, published studies demonstrating their effective industrial field usage in grain stores and food production facilities are scarce. This review shows that the current problems associated with the application of some natural botanical insecticides (e.g., sorption, stability, field efficacy, and smell) to some extent echo problems that were frequently encountered and addressed almost 100 years ago during the transition from ancient to modern classical chemical pest control methods.

Comparative mitochondrial genomics of five Dermestid beetles (Coleoptera: Dermestidae) and its implications for phylogeny.

Dermestid beetles (Coleoptera: Dermestidae) are important pests of various stored products, posing potential threats to international trade. Their detailed characterization on molecular basis is a pre-requisite for proper identification and for understanding of their phylogenetic relationships. In this work, the whole mitochondrial genomes (mitogenomes) of Trogoderma granarium, Dermestes lardarius, D. ater, Attagenus augustatus augustatus and Attagenus unicolor japonicus were firstly sequenced to update the database using the next-generation sequencing technique. Based on the selected model species, a comparative analysis of four Dermestidae genera was performed. The mitochondrial genomes of these five species above showed high similarity in nucleotide composition, base composition and gene order, including 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs) and a non-coding control region, which was similar to most of Coleoptera species. The phylogenetic analysis based on the PCGs and two rRNAs indicated that the relationships within Dermestidae were reconstructed as (((Trogoderma + Anthrenus) + Attagenus) + Dermestes) using both Maximum Likelihood (ML) and Bayesian Inference (BI) analysis. However, more mitogenomes should be sequenced to obtain a more holistic view of the whole family. This study not only showed the mitogenomes of five Dermestidae species and their high conservativeness, but also discussed its implications for reconstructing a more comprehensive phylogeny of dermestids.

Efficacy of rodenticide baits with decreased concentrations of brodifacoum: Validation of the impact of the new EU anticoagulant regulation.

Anticoagulants are the most frequently used rodenticides at the global scale. Because of their persistency, bioaccumulation and potential for secondary intoxication, they have faced increasing legislative regulations. Recently, the European Union Regulation (EU) 2016/1179 resulted in the production and application of rodenticides with nearly half dose (<30 ppm) of anticoagulants. However, published data on the biological efficacy of rodenticides with decreased doses are scarce in the EU. Therefore, this work compared the efficacy of the original high-dose (50 ppm) and new low-dose (25 ppm) brodifacoum-based baits in the offspring of wild-caught house mice (Mus musculus L.). In the no-choice laboratory feeding tests, 100% animals died in all treated groups and 0% died in the control groups. The achieved time to death did not differ between the original and low-dose baits across both types of feeding trials/regimes. The low-dose baits (25 ppm) were consequently tested under field conditions in two populations showing 95.7% and 99.8% efficacy. The obtained results highlighted the good efficacy of the new baits based on low-dose brodifacoum in non-resistant mouse populations. However, further validation is required regarding the remaining anticoagulant compounds and resistant rodent populations.

Minimal Thermal Requirements for Development and Activity of Stored Product and Food Industry Pests (Acari, Coleoptera, Lepidoptera, Psocoptera, Diptera and Blattodea): A Review.

Low temperatures play an important role in arthropods because they affect both the individual and population development of all physiological and behavioural activities. Manipulation with low temperatures is a primary nonchemical pest control method. For stored product and food industry practitioners, a knowledge of pest thermal requirements, in particular threshold temperatures at which development and other activities of a particular pest species cease, is of crucial importance. This review presents summary data regarding the lower temperature thresholds of 121 species of stored product and food industry pests from six arthropod taxa (Acari, Coleoptera, Lepidoptera, Psocoptera, Diptera, and Blattodea). In particular, this review collected and summarized information regarding the lower development thresholds, lower population thresholds, lower acoustic or respiratory thresholds, lower walking and flying thresholds and lower trap capture thresholds for flying and walking arthropods. The average lower development threshold (LDT) differed among orders: the lowest was reported for Acari (6.8 °C) and Diptera (8.1 °C), followed by Lepidoptera (11.3 °C) and Psocoptera (13.8 °C), and the highest was reported for Coleoptera (14 °C) and Blattodea (15 °C). An exclusion-function was established showing the percentage of pest species (n = 112) that were developmentally suppressed (excluded) due to temperatures reaching the LDT in the range of decreasing temperatures from 25 °C to 0 °C. We scaled various temperature thresholds from the lowest to highest temperature as follows: the walking threshold, the trap capture threshold for walking insects, the lower development threshold, lower population threshold, lower flying threshold and the lower trap capture threshold for flying pests. Important pest species were identified for which information regarding the lower temperature threshold is missing, or for which the information is too variable and should be refined in future research.

First Case of Dual Size Asymmetry in an Identical Arthropod Organ: Different Asymmetries of the Combative (Sexual) and Cutting (Non-Sexual) Parts of Mandibles in the Horned Stored-Product Beetle Gnatocerus cornutus (Fabricius, 1798).

Although it is known that separate insect body structures may be asymmetrical within one species, the different functional asymmetries within a single organ as a result of differential selective regimes have not been described. Based on microscopic measurements and SEM photography, we examined the size, shape and asymmetry of the mandibular structures of males and females of the sexually dimorphic broad-horned flour beetle, Gnatocerus cornutus (Tenebrionidae, Coleoptera). It was found that sexual dimorphism only manifests in certain outgrowth parts (horns) of male mandibles, while the remaining cutting parts of the mandibles hold identical morphologies for both sexes. A more interesting finding-since this is the first published case of dual functionally selected asymmetry in an identical arthropod organ-was that the cutting part of the male mandible exhibited directional asymmetry, whereas the outgrowth horn part of the mandible showed a high degree of symmetry. Moreover, there was no relationship between the size and asymmetry of horns. The results indicate different regulatory mechanisms of sexually selected combative horns and the food-functional, more conservative (constrained by hard food and adult long life) cutting parts of mandibles.

Molecular Identification of ten species of stored-product psocids through microarray method based on ITS2 rDNA.

Stored-product psocids (Psocoptera: Liposcelididae) are cosmopolitan storage pests that can damage stored products and cause serious economic loss. However, because of the body size (~1 mm) of eggs, nymphs, and adults, morphological identification of most stored-product psocids is difficult and hampers effective identification. In this study, 10 economically important stored-product Liposcelis spp. psocids (Liposcelis brunnea, L. entomophila, L. decolor, L. pearmani, L. rufa, L.mendax, L. bostrychophila, L. corrodens, L. paeta, and L. tricolor) were collected from 25 geographic locations in 3 countries (China, Czech Republic, and the United States). Ten species-specific probes for identifying these 10 psocid species were designed based on ITS2 sequences. The microarray method and reaction system were optimized. Specificity of each of the ten probes was tested, and all probes were found suitable for use in identification of the respective10 Liposcelis spp. psocids at 66 °C. This method was also used to identify an unknown psocid species collected in Taian, China. This work has contributed to the development of a molecular identification method for stored-product psocids, and can provide technical support not only to facilitate identification of intercepted samples in relation to plant quarantine, but also for use in insect pest monitoring.

Differences in the Bacterial Community of Laboratory and Wild Populations of the Predatory Mite Cheyletus eruditus (Acarina: Cheyletidae) and Bacteria Transmission From Its Prey Acarus siro (Acari: Acaridae).

The parthenogenetic predatory mite Cheyletus eruditus (Schrank, 1781) is used for biological control against mite pests produced as CHEYLETIN. Although there is evidence that bacteria are mainly responsible for parthenogeny in several species of predatory mites, the description of association between C. eruditus the specific and parasitic or symbiotic bacteria is still missing. We analyzed the bacterial communities of the predator, C. eruditus , and its prey, Acarus siro L. The 16S rRNA gene was amplified, cloned, and sequenced. The selected bacterial taxa were confirmed by amplification of isolated DNA with taxon-specific primers. The 16S rRNA gene sequences from the predatory and prey mites formed a total of 20 different bacterial taxa. Of these taxa, the predator and prey shared four taxa, six taxa were specific for the predatory, and 10 taxa for the prey mites. Cardinium - and Bartonella -like bacteria were found in both mite species. The reproductive parasite Wolbachia was found only in the predatory mite, and A. siro hosted Solitalea -like (Sphingobacteriales) bacteria that were not detected in C. eruditus . We focused on Cardinium occurrence in the field samples of C. eruditus. Using Cardinium -specific primers, 128 clones were obtained. Cardinium was found in seven field samples of C. eruditus as well as in the laboratory population that was used to produce CHEYLETIN. Phylogenetic analysis of the Cardinium clones identified three separate clusters: two clusters showed high similarity to the Cardinium sequences from astigmatid mites, and one cluster contained only the clones from C. eruditus . Sequences of both Cardinium and Wolbachia were found in the both adults and eggs of C. eruditus , indicating maternal transfer of these endosymbiotic bacteria.

The effect of stored barley cultivars, temperature and humidity on population increase of Acarus siro, Lepidoglyphus destructor and Tyrophagus putrescentiae.

The rate of population increase of three mite species, Acarus siro (L.), Lepidoglyphus destructor (Schrank) and Tyrophagus putrescentiae (Schrank), was studied on various types of barley and at various combinations of temperature and humidity. The mites were added into the chambers and incubated for 21 days on seven different kinds of barley coming from four sites, including six cultivars and a mixture. The population increase of all species was higher on the mixture than on any other cultivar, except for Sebastian and Calgary. The increase of mites was studied at constant temperatures ranging from 5 to 35 °C and relative humidity (RH) ranging from 50 to 90 %. Positive rate of increase was found above 70 % RH for all species. The optimal humidity was at 85 % RH for A. siro and L. destructor and at 90 % RH for T. putrescentiae. As concerns the temperature, positive rate of increase was found at temperatures higher than 10, 15 and 20 °C for A. siro, L. destructor and T. putrescentiae, respectively. The temperature optima were at 23, 25, and 30 °C for A. siro, L. destructor and T. putrescentiae, respectively. Model estimated on laboratory data was then fitted to temperature and humidity records from August to November in the Czech grain store. Estimated population rate of increase was rarely positive: for A. siro it was for 24 %, for L. destructor for only 1 % and for T. putrescentiae for only 7 % days of the study period. It is concluded that in the climatic conditions of the Czech Republic the population increase of three mite pests is negligible during autumn and winter.

Production of UV-light-detectable faeces from house mice (Mus musculus domesticus) after consumption of encapsulated fluorescent pigment in monitoring bait.

BACKGROUND: The authors investigated whether fluorescent pigment in thermoset melamine microcapsules incorporated into monitoring baits would be excreted in the faeces of wild house mice in a quantity and intensity that would be detectable by a human observer. RESULTS: Experimental mice produced 24-116 UV-visible faecal pellets per 24 h; the mean dry weight was 582 mg. The number and weight of the faeces was independent of mouse sex and weight. The defecation of UV-visible faeces began at 2-3 h, peaked at 5-8 h and was complete at 17 h after bait ingestion. The detectability of the highly fluorescent faecal pellets using a small UV flashlight approached 100%, and no false positives were recorded. CONCLUSION: The tested formulation is of significant value for rodent pest monitoring because faeces that are highly visible by UV light are produced for 15 h by mice after ingestion, and their detection is easy and unambiguous.

The influence of environmental temperature and humidity on temporal decomposition of cockroach allergens Bla g 1 and Bla g 2 in feces.

The aim of the study was to establish a model of the environmental fate of German cockroach (Blattella germanica L.) allergens Bla g 1 and Bla g 2 in feces under controlled and field conditions. Temporal decline (3, 6, and 9 mo) of allergens Bla g 1 and Bla g 2 in the feces protected from cleaning was measured under laboratory and experimental household conditions. The influence of environmental temperature (15, 20, 25, 30, and 35 degrees C) and moisture (53, 75, 85, and 100% RH) on allergen degradation was estimated for 3, 6, and 9 mo. Bla g 1 was more stable than Bla g 2 and the proteins. The proteins and Bla g 2 contents were correlated negatively with the decomposition time; Bla g 1 was not. However, when the content of Bla g 1 in control and exposed tubes was compared, the decrease after exposure was significant at exposure in 35 degrees C, 53 and 100% RH. In laboratory, the shortest half-life (16-38 d) of Bla g 2 was at high temperature and humidity (100% RH at 35 degrees C), whereas the longest half-life (340 d) was at 25 degrees C and 85% RH. In the apartment, the half-life was 406 d. The results indicate that Bla g 1 and Bla g 2 allergens can persist in feces for several months under usual household humidity and temperature.

Brief exposure of Blattella germanica (Blattodea) to insecticides formulated in various microcapsule sizes and applied on porous and non-porous surfaces.

BACKGROUND: The authors explored how microcapsule size and brief exposure affected the bioavailability of five microencapsulated insecticide formulations, chlorpyrifos 23.1 g L(-1) CS (Detmol-PRO), chlorpyrifos 20 g L(-1) CS (Empire 20), fenitrothion 20 g L(-1) CS (Detmol-Mic), cyphenothrin 10 g L(-1) CS (Detmol-CAP) and diazinon 30 g L(-1) CS (Diacap), to Blattella germanica L. on porous and non-porous surfaces. The hypothesis was tested that microencapsulated (CS) insecticides comprising larger microcapsules show higher efficacy on porous surfaces than formulations with smaller microcapsules. RESULTS: Brief exposure was accomplished by allowing B. germanica to cross a 0.3 m insecticide barrier in 30 s (1.01 cm s(-1)). Such short exposure did not lead to 100% mortality in any formulation or surface tested. Significant differences in bioavailability on the porous and the non-porous surfaces were found: the largest difference was observed in Empire 20 and Detmol CAP, while bioavailability of Detmol MIC did not differ on porous and non-porous surfaces. Comparison of their microcapsule size spectra revealed that formulations containing larger microcapsules had higher efficacy on porous surfaces than formulations with smaller microcapsules. In order to explain the difference in efficacy, the variance of microcapsule sizes was regressed on the efficacy ratio on porous versus non-porous surfaces. Although negative correlation was evident between size of capsules and the efficacy ratio on porous and non-porous surfaces, the difference in the slope parameter was not statistically significant. CONCLUSION: Brief contact of B. germanica with insecticide spray residues, which is common in barrier treatment, may lead to low efficacy, especially on porous surfaces. The latter should be preferably treated with CS insecticides containing a fraction with large capsules. In addition to the size of the microcapsules, the role of other factors, such as wall capsule thickness and chemical composition, on CS insecticide activity on various surfaces should be examined in future work.