Trehalose 6-phosphate synthase gene rdtps1 contributes to thermal acclimation in Rhyzopertha dominica.

BACKGROUND: The lesser grain borer (Rhyzopertha dominica), a worldwide primary pest of stored grain, causes serious economic losses and threatens stored food safety. R. dominica can respond to changes in temperature, especially the adaptability to heat. In this study, transcriptome analysis of R. dominica exposed to different temperatures was performed to elucidate differences in gene expression and the underling molecular mechanism. RESULTS: Isoform-sequencing generated 17,721,200 raw reads and yielded 20,416 full-length transcripts. A total of 18,880 (92.48%) transcripts were annotated. We extracted RNA from R. dominica reared at 5 °C (cold stress), 15 °C (cold stress), 27 °C (ambient temperature) and 40 °C (heat stress) for RNA-seq. Compared to those of control insects reared at 27 °C, 119, 342, and 875 differentially expressed genes (DEGs) were identified at 5 °C, 15 °C, and 40 °C, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that pathways associated with "fatty acid metabolism", "fatty acid biosynthesis", "AMPK signaling pathway", "neuroactive ligand receptor interaction", and "longevity regulating pathway-multiple species" were significantly enriched. The functional annotation revealed that the genes encoding heat shock proteins (HSPs), fatty acid synthase (FAS), phospholipases (PLA), trehalose transporter (TPST), trehalose 6-phosphate synthase (TPS), and vitellogenin (Vg) were most likely involved in temperature regulation, which was also validated by RT-qPCR. Seven candidate genes (rdhsp1, rdfas1, rdpla1, rdtpst1, rdtps1, rdvg1, and rdP450) were silenced in the RNA interference (RNAi) assay. RNAi of each candidate gene suggested that inhibiting rdtps1 expression significantly decreased the trehalose level and survival rate of R. dominica at 40 °C. CONCLUSIONS: These results indicated that trehalose contributes to the high temperature resistance of R. dominica. Our study elucidates the molecular mechanisms underlying heat tolerance and provides a potential target for the pest management in R. dominica.

Stability evaluation of candidate reference genes for real-time qPCR normalization in Rhyzopertha dominica (Coleoptera: Bostrycidae).

Rhyzopertha dominica is a serious stored grain insect pest around the world. Real-time quantitative polymerase chain reaction (RT-qPCR) is a widely used experimental method in molecular biology for detecting the expression of target genes. As appropriate reference genes are essential for normalizing gene expression, the selection of suitable reference genes is the basis of RT-qPCR experiments. In this study, the expression profiles of 7 candidate reference genes of rps3, rps6, rps13, actin, gadph, tubulin, and 18S rRNA were analyzed under 4 different experimental conditions. The expression stability of candidate genes was evaluated using the ΔCt, GeNorm, BestKeeper, NormFinder, and RefFinder methods. The results revealed that different reference genes were suitable for various experiments. Specifically, rps3 and rps6 were appropriate for the developmental stages and all samples: 18S rRNA and rps13 for temperature-related experiments, actin and rps6 for sex-related experiments, and rps6 and gadph for starvation stress. Our results lay essential groundwork for the normalization of RT-qPCR analyses and contribute to genomic and gene functional research of R. dominica.

Regulation of Aquaporin Prip Expression and Its Physiological Function in Rhyzopertha dominica (Coleoptera: Bostrichidae).

Rhyzopertha dominica Prip (RdPrip) cDNA was cloned (GenBank accession no. OK318454), and the encoded 276-amino-acid protein indicated the typical aquaporin structure, including six transmembrane regions and two NPA motifs. The developmental and tissue profiles of RdPrip transcription were determined. RdPrip was highly transcribed in female adults, followed by larvae, pupae, and male adults. The transcriptional expression levels of RdPrip were significantly high in the ovary and hindgut (including cryptonephridial systems) compared with the foregut, testis, midgut, and Malpighian tubules. Knockdown of RdPrip in female adults did not decrease fecundity, but significantly decreased the hatching rate of eggs laid by the females. The results suggest that RdPrip functions in embryonic development, not in egg formation. In addition, the transcriptional expression level of RdPrip was lower in the spinosad-resistant strain than in the susceptible one, and the resistant strain produced fewer progeny than the susceptible strain did. These studies support the functional role of RdPrip in female reproduction. The absence of significant mortality reduction in the R. dominica exposed to spinosad after RdPrip RNAi suggests that other aquaporins that were not knocked down may exist for the excretion of metabolized pesticides.

Ozone reduces lifespan and alters gene expression profiles in Rhyzopertha dominica (Fabricius).

Rhyzopertha dominica is one of the most important stored grain pests that seriously damage rice and wheat. At present, the method of controlling stored grain pests mainly relies on insecticide fumigation. However, the excessive use of pesticides not only leaves pesticide residues, with harmful effects on human health and the environment, but also induces insect resistance. Ozone is a strong oxidant with the characteristics of easy decomposition and without residue. Although ozone has been widely used in the food industry in recent years, research on the control of stored grain pests is limited. In this research, we used ozone treatment to control R. dominica adults and explore the molecular mechanisms that affect them. Here, we found that ozone treatment on R. dominica adults could decrease life span and increase malondialdehyde (MDA) content, as well as reduce activity of total superoxide dismutase (SOD) and catalase (CAT). Using RNA-seq technology, we identified 641 genes that were differentially expressed between ozone-treated and control R. dominica adults [fold-change of ≥ 2 (q-value < 5%)]. When comparing ozone treatment with control R. dominica adults, 330 genes were significantly upregulated and 311 were downregulated. RT-qPCR confirmed that 11 genes were differentially expressed in ozone-treated and control R. dominica adults. These genes were involved in insect cuticle protein and antioxidant system. This research showed that ozone treatment could reduce the lifespan of R. dominica through antioxidant system. It is an environmentally benign method for the control of stored grain pests and has great development potential.

Promising Insecticidal Efficiency of Essential Oils Isolated from Four Cultivated Eucalyptus Species in Iran against the Lesser Grain Borer, Rhyzopertha dominica (F.).

The lesser grain borer, Rhyzopertha dominica (F.), causes damage to stored grains resulting in both quantitative and qualitative losses. The use of synthetic fumigants in the management of stored-product pests resulted in undesirable side effects such as environmental contamination and threat to human and animal health. In this study, the lethal and sublethal effects of essential oils from four Eucalyptus species, E. microtheca, E. procera, E. spatulata, and E. torquata were studied against R. dominica adults. Gas chromatographic-mass spectral analysis of the essential oils was carried out, in which terpenes such as 1,8-cineole and globulol were abundant in essential oils. The pest was susceptible to the fumigation of the essential oils and, considering concentrations and exposure times (24, 48, and 72 h), had significant effects on the pest mortality. The total protein, glycogen, and lipid contents and digestive amylolytic and proteolytic activities of the adults treated with tested essential oils were reduced. The consumption index, relative consumption rate, and relative growth rate were also reduced in the treated adults. According to the insecticidal effects on the adults of R. dominica, the essential oils of E. microtheca, E. procera, E. spatulata, and E. torquata can be candidates for further investigations as grain protectant agents.

Chemical Profiles and Insecticidal Potential of Essential Oils Isolated from Four Thymus Species against Rhyzopertha dominica (F.).

Although chemical pesticides have been efficiently used to manage insect pest, their overuse has led to environmental contamination and threats to human health, enticing researchers to introduce eco-friendly and effective agents. In this study, the insecticidal effectiveness of essential oils isolated from Thymus species, including T. eriocalyx, T. kotschyanus, T. fallax, and T. vulgaris, was evaluated against the adults of Rhyzopertha dominica. The terpenes p-cymene, 1,8-cineole, linalool, α-terpineol, and carvacrol were the prominent compounds in the hydrodistilled essential oils. All essential oils produced significant fumigant at 24, 48, and 72-exposure times. The energy reserves protein by all essential oils, glycogen by T. kotschyanus and T. vulgaris, and lipid by T. fallax and T. vulgaris were significantly decreased compared to control. All essential oils except T. vulgaris affected the amylolytic and proteolytic activity of the pest. The pest increased the α- and ß-esterase enzyme activity in response to the essential oils. Nutritional indices of adults were also affected by essential oils, in which feeding deterrence index was calculated from 20.41% to 61.11%. Accordingly, based on lethal and extensive sub-lethal insecticidal activities, T. eriocalyx, T. kotschyanus, T. fallax, and T. vulgaris essential oils can be considered as efficient agents for R. dominica management.

Efficiency of a Novel Light-Emitting Diode (LED) Trap for Trapping Rhyzopertha dominica (Coleoptera: Bostrichidae) in Paddy Rice Storehouses.

The lesser grain borer Rhyzopertha dominica is the major pest of stored paddy rice globally, including in Taiwan. It has strong phototaxis and is good at flying, suitable for developing a light-trapping method to monitor and control it. In the present study, a wavelength of light-emitting diodes (LEDs), i.e., 373 nm, was determined to be the most efficient to trap R. dominica using a dodecagon maze. Accordingly, an LED trap, named the Taiwan Agricultural Research Institute-LED (TARI-LED) trap, was invented, which comprised LEDs of two distinct wavelengths (373 and 408 nm), a wavelength switch, a suction fan, and an insect collector. The trapping efficiency was assessed in a 4-m3 laboratory arena and two paddy rice storehouses. An initial assessment was performed in the laboratory arena and showed that the TARI-LED trap with 373-nm wavelength for R. dominica rapidly increased in the first 30 min, reaching the highest trapping rate (68.5%) after 3 h. In addition, no significant difference was observed between the suction fan turned on or off. The field tests showed that the 373-nm wavelength had the highest effectiveness for trapping R. dominica in the two paddy rice storehouses, and no significant difference was observed in the number of R. dominica trapped by the 373-nm TARI-LED trap or the CDC-UV light trap. In conclusion, our TARI-LED trap 373 nm exhibited high efficiency in trapping R. dominica in paddy rice storehouses. Moreover, a suction fan-free design should benefit long-term and safe use in paddy rice storehouses trapping R. dominica.

The Genome of Rhyzopertha dominica (Fab.) (Coleoptera: Bostrichidae): Adaptation for Success.

The lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), is a major global pest of cereal grains. Infestations are difficult to control as larvae feed inside grain kernels, and many populations are resistant to both contact insecticides and fumigants. We sequenced the genome of R. dominica to identify genes responsible for important biological functions and develop more targeted and efficacious management strategies. The genome was assembled from long read sequencing and long-range scaffolding technologies. The genome assembly is 479.1 Mb, close to the predicted genome size of 480.4 Mb by flow cytometry. This assembly is among the most contiguous beetle assemblies published to date, with 139 scaffolds, an N50 of 53.6 Mb, and L50 of 4, indicating chromosome-scale scaffolds. Predicted genes from biologically relevant groups were manually annotated using transcriptome data from adults and different larval tissues to guide annotation. The expansion of carbohydrase and serine peptidase genes suggest that they combine to enable efficient digestion of cereal proteins. A reduction in the copy number of several detoxification gene families relative to other coleopterans may reflect the low selective pressure on these genes in an insect that spends most of its life feeding internally. Chemoreceptor genes contain elevated numbers of pseudogenes for odorant receptors that also may be related to the recent ontogenetic shift of R. dominica to a diet consisting primarily of stored grains. Analysis of repetitive sequences will further define the evolution of bostrichid beetles compared to other species. The data overall contribute significantly to coleopteran genetic research.

Variants in the Mitochondrial Genome Sequence of Rhyzopertha dominica (Fabricius) (Coleoptera: Bostrycidae).

The lesser grain borer, Rhyzopertha dominica, is a coleopteran pest of stored grains and is mainly controlled by phosphine fumigation, but the increase in phosphine-resistant populations threatens efficacy. Some phosphine-resistant insects have reduced respiration, and thus studying the mitochondrial genome may provide additional information regarding resistance. Genomic DNA from an inbred laboratory strain of R. dominica was extracted and sequenced with both short (Illumina) and long (Pacific Biosciences) read technologies for whole genome sequence assembly and annotation. Short read sequences were assembled and annotated by open software to identify mitochondrial sequences, and the assembled sequence was manually annotated and verified by long read sequences. The mitochondrial genome sequence for R. dominica had a total length of 15,724 bp and encoded 22 trna genes, 2 rRNA genes, 13 protein coding genes (7 nad subunits, 3 cox, 2 atp, and 1 cytB), flanked by a long control region. We compared our predicted mitochondrial genome to that of another from a R. dominica strain from Jingziguan (China). While there was mostly agreement between the two assemblies, key differences will be further examined to determine if mutations in populations are related to insecticide control pressure, mainly that of phosphine. Differences in sequence data, assembly, and annotation also may result in different genome interpretations.

Transcriptomic analysis of s-methoprene resistance in the lesser grain borer, Rhyzopertha dominica, and evaluation of piperonyl butoxide as a resistance breaker.

BACKGROUND: The lesser grain borer, Rhyzopertha dominica is a serious pest of stored grains. Fumigation and contact insecticides play a major role in managing this pest globally. While insects are developing genetic resistance to chemicals, hormonal analogues such as s-methoprene play a key role in reducing general pest pressure as well as managing pest populations that are resistant to fumigants and neurotoxic contact insecticides. However, resistance to s-methoprene has been reported in R. dominica with some reports showing a remarkable high resistance, questioning the use of this compound and other related analogues in grain protection. The current study attempts to identify possible molecular mechanisms that contribute in resistance to s-methoprene in R. dominica. RESULTS: Transcriptome analysis of resistant and susceptible strains of this pest species identified a set of differentially expressed genes related to cytochrome P450s, indicating their potential role in resistance to s-methoprene. Laboratory bioassays were performed with s-methoprene treated wheat grains in presence and absence of piperonyl butoxide (PBO), a cytochrome P450 inhibitor. The results indicate that PBO, when applied alone, at least at the concentration tested here, had no effect on R. dominica adult emergence, but has a clear synergistic effect to s-methoprene. The number of produced progeny decreased in presence of the inhibitor, especially in the resistant strain. In addition, we also identified CYP complement (CYPome) of R. dominica, annotated and analysed phylogenetically, to understand the evolutionary relationships with other species. CONCLUSIONS: The information generated in current study suggest that PBO can effectively be used to break resistance to s-methoprene in R. dominica.

Toxicity and Sublethal Effects of Phthalides Analogs to Rhyzopertha dominica.

Phthalides and their precursors have demonstrated a large variety of biological activities. Eighteen phthalides were synthesized and tested on the stored grain pest Rhyzopertha dominica. In the screening bioassay, compounds rac-(2R,2aS,4R,4aS,6aR,6bS,7R)-7-bromohexahydro-2,4-methano-1,6-dioxacyclopenta[cd]pentalen-5(2H)-one (15) and rac-(3R,3aR,4R,7S,7aS)-3-(propan-2-yloxy)hexahydro-4,7-methano-2-benzofuran-1(3H)-one (17) showed mortality similar to the commercial insecticide, Bifenthrin® (≥90 %). The time (LT50 ) and dose (LD50 ) necessary to kill 50 % of the R. dominica population were determined for the most efficacious phthalides 15 and 17. Compound 15 presented the lowest LD50 (1.97 µg g-1 ), being four times more toxic than Bifenthrin® (LD50 =9.11 µg g-1 ). Both compounds presented an LT50 value equal to 24 h. When applied at a sublethal dose, both phthalides (especially compound 15), reduced the emergence of the first progeny of R. dominica. These findings highlight the potential of phthalides 15 and 17 as precursors for the development of insecticides for R. dominica control.

Characterization of the complete mitochondrial genome of lesser grain borer Rhyzopertha dominica Fabricius (Insecta: Coleoptera: Bostrichidae) from Jingziguan.

The lesser grain borer, Rhyzopertha dominica (Fabricius) is a primary pest of starch-containing stored products worldwide. Here, we report characterization of mitogenome of R. dominica and its phylogenetic position. Rhyzopertha dominica complete mitochondrial genome (GenBank accession number MN527959) from Jingziguan town consisted of a circular DNA molecule of 15,862 bp (with 74.36% A + T content). The mitogenome comprised of 13 protein-coding genes (PCGs), and 22 tRNA and two rRNA genes. PCGs had typical ATN (Met) initiation codons and were terminated by typical TAN stop codons.

Synthesis, insecticidal activity, and phytotoxicity of novel chiral amides.

BACKGROUND: The lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae), is an important pest of stored grains worldwide. Chemical control is the main method used to manage this pest, but the continuous use of insecticides can lead to the selection of resistant R. dominica strains. Thus, there is a constant demand for the development of new insecticide molecules. This study describes the synthesis of 14 chiral amides and evaluation of their insecticidal activity against R. dominica. Their phytotoxicity to wheat (Triticum sativum) seeds is also evaluated. RESULTS: In the screening assay, compounds 8i and 8j caused 100% and 87% mortality of R. dominica. These values did not differ from the mortality caused by Bifenthrin® (75%). Amide 8i presented similar toxicity (LD50  = 27.98 µmol g-1 , CI95  = 25.14-30.71) and speed of action (LT50  = 22 h, CI95  = 19.34-24.66) to amide 8j (LD50  = 29.37 µmol g-1 , CI95  = 27.43-31.09, and LT50  = 19 h, CI95  = 17.05-20.95) against the pest. Both amides inhibited less than 44% of wheat growth. CONCLUSION: Among the tested amides, only 8i and 8j were effective in R. dominica control and presented no considerable phytotoxicity towards wheat seeds. Therefore, these amides are promising as insecticides for the management of R. dominica. © 2018 Society of Chemical Industry.

Nicotinic acetylcholine receptor subunit α6 associated with spinosad resistance in Rhyzopertha dominica (Coleoptera: Bostrichidae).

The lesser grain borer, Rhyzopertha dominica, which is a primary pest of stored products, breaks up whole grains and makes them susceptible to secondary infestation by other pests. Insecticide application is the main control measure against this borer. A resistant strain of R. dominica against the insecticide, spinosad, was selected in the laboratory. The full-length cDNA of the target site of spinosad, nicotinic acetylcholine receptor subunit α6, from R. dominica (Rdα6) was cloned and analyzed using reverse transcription PCR and rapid amplification of cDNA ends. The complete 2133-bp cDNA contains the open reading frame of 1497 bp encoding a 498-amino-acid protein. There are four predicted transmembrane (TM) regions, and six extracellular ligand-binding sites at the N-terminus, upstream from the first TM in Rdα6. Three mutations have been found in the resistant strain compared with the susceptible one: (1) a 181-bp fragment truncated at the N-terminus, resulting in the appearance of a premature stop codon, (2) one missing bp at the position 997, causing a frame-shift mutation, and (3) an 87-bp fragment truncated in the TM2 region. In addition, real-time quantitative PCR was applied to detect the transcriptional expression of Rdα6 in both the susceptible and resistant strains. The results indicated that the expression of Rdα6 was significantly lower in then resistant strain than in susceptible one. In conclusion, mutation of Rdα6 may cause R. dominica resistant to spinosad due to target site insensitivity.

Novel bacteriocyte-associated pleomorphic symbiont of the grain pest beetle Rhyzopertha dominica (Coleoptera: Bostrichidae).

BACKGROUND: The lesser grain borer Rhyzopertha dominica (Coleoptera: Bostrichidae) is a stored-product pest beetle. Early histological studies dating back to 1930s have reported that R. dominica and other bostrichid species possess a pair of oval symbiotic organs, called the bacteriomes, in which the cytoplasm is densely populated by pleomorphic symbiotic bacteria of peculiar rosette-like shape. However, the microbiological nature of the symbiont has remained elusive. RESULTS: Here we investigated the bacterial symbiont of R. dominica using modern molecular, histological, and microscopic techniques. Whole-mount fluorescence in situ hybridization specifically targeting symbiotic bacteria consistently detected paired bacteriomes, in which the cytoplasm was full of pleomorphic bacterial cells, in the abdomen of adults, pupae and larvae, confirming previous histological descriptions. Molecular phylogenetic analysis identified the symbiont as a member of the Bacteroidetes, in which the symbiont constituted a distinct bacterial lineage allied to a variety of insect-associated endosymbiont clades, including Uzinura of diaspidid scales, Walczuchella of giant scales, Brownia of root mealybugs, Sulcia of diverse hemipterans, and Blattabacterium of roaches. The symbiont gene exhibited markedly AT-biased nucleotide composition and significantly accelerated molecular evolution, suggesting degenerative evolution of the symbiont genome. The symbiotic bacteria were detected in oocytes and embryos, confirming continuous host-symbiont association and vertical symbiont transmission in the host life cycle. CONCLUSIONS: We demonstrate that the symbiont of R. dominica constitutes a novel bacterial lineage in the Bacteroidetes. We propose that reductive evolution of the symbiont genome may be relevant to the amorphous morphology of the bacterial cells via disruption of genes involved in cell wall synthesis and cell division. Genomic and functional aspects of the host-symbiont relationship deserve future studies.

Analysis of volatiles from stored wheat and Rhyzopertha dominica (F.) with solid phase microextraction-gas chromatography mass spectrometry.

BACKGROUND: Volatile organic compounds (VOCs) contribute significantly to food flavour and can be used as indicators of quality, age of storage, and hygiene condition of stored products. The VOCs in the headspace of three different samples - healthy wheat, Rhyzopertha dominica, and wheat with R. dominica - were analysed at 25°C by solid phase micro-extraction (SPME) coupled with gas chromatography-flame ionisation detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). All the experimental conditions were kept consistent except a polar column and a non-polar column were used to assess the differences in volatile fingerprints. RESULTS: A total of 114 volatiles were identified by both the polar and non-polar columns, of which 48 were specific to one of the three samples tested. The volatiles were mainly carbonyl chemical compounds such as aldehydes, ketones and alcohols. GC-MS results showed slightly more VOCs were identified from the polar column. The total number for the three samples was 43 from the polar column compared to 39 from the non-polar column. Conversely, 30 VOCs unique to a given sample were identified from the non-polar column compared to 18 from the polar column. CONCLUSION: The use of both polar and non-polar columns is essential to capture the full range of VOCs produced by the three specific sample types investigated. The data can form the basis of enquiry into the relationship between storage and grain quality, and insect infestation and grain quality by observing the impact that these circumstances have on the production of volatile organic compounds.

Effect of temperature and grain type on the long-term persistence and efficacy of s-methoprene in controlling Rhyzopertha dominica.

BACKGROUND: The efficacy of methoprene can vary with surface substrates, application methods and environmental conditions. The objectives of the present study were to examine the effects of temperature and grain type on the long-term persistence and efficacy of methoprene in controlling Rhyzopertha dominica from Guangzhou, China. RESULTS: Methoprene applied at 1 mg kg(-1) caused >90% suppression of F1 adult progeny of R. dominica for 150-270 days. Temperature and grain type both influenced the long-term persistence of methoprene. Overall multivariate analysis of variance showed that the order of progeny reduction at different temperatures (°C) was: 24 > 28 > 32 > 36; the order of the progeny reduction on the different grains was: paddy > wheat and maize. CONCLUSION: The results of our experiments show the maximum effect of methoprene for R. dominica control at 24 °C on paddy.

Spread of phosphine resistance among brazilian populations of three species of stored product insects

The resistance to fumigant insecticides in stored-products insects is often recorded. Several factors influence the evolution of insecticide resistance. Among these, the frequency of applications and the migration of resistant populations are of primary importance for the stored-product insects. The aim of this study was to characterize the spectrum and investigate the status of phosphine resistance in Brazil, in 13 populations of the Coleoptera Tribolium castaneum Herbst (Tenebrionidae), ten populations of Rhyzopertha dominica (Fabr.) (Bostrichidae), and eight populations of Oryzaephilus surinamensis (L.) (Silvanidae). The pattern of resistance dispersion in the populations of these species was also verified. The bioassays for the detection of phosphine resistance followed the FAO standard method. To test the influence of migration in the evolution of the phosphine resistance, the difference of mortality in the discriminating concentration and the geographical distance among each pair wise combination of collection sites were correlated. None of the populations exhibited mortality above 90 percent in the discriminating concentration, for the three species. Mortality in the discriminating concentration increased with the geographical distance for R.dominica and O.surinamensis. However, no significant linear response was observed among the variables for T.castaneum populations. These results suggest that the dispersion of insects and the local selection are relevant in the evolution of the phosphine resistance in populations of R.dominica and O.surinamensis. In contrast, grain trade and local selection are probably the factors that determine the evolution of the phosphine resistance in populations of T. castaneum.