Genetics of resistance to phosphine in Rhyzopertha dominica (Coleoptera: Bostrichidae)

The inheritance of resistance to phosphine was studied in two strains of the lesser grain borer, Rhyzopertha dominica (F.), labeled 'Weak-R' and 'Strong-R'. These strains were purified versions of field-selected populations collected in Queensland, Australia. Weak-R and Strong-R were, respectively, 23.4 times (20-h exposure) and 600 times (48-h exposure) resistant to phosphine compared with a reference susceptible strain (S-strain). Each -R strain was crossed with the S-strain and the response to phosphine was measured in their respective F1, F2, and F1-backcross (F1-BC) progenies. Data from testing of reciprocal F1 progeny indicated that resistance in Weak-R was autosomal and incompletely recessive with a degree of dominance -0.96. Modified chi-square analysis and contingency analysis of the observed response to phosphine of F1-BC and F2 progenies rejected the hypothesis of single gene inheritance of resistance. Analysis of the response of the F1, F2, and F1-BC progeny from the Strong-R x S-strain cross also rejected the null hypothesis for single gene resistance. Resistance in the Strong-R strain was autosomal and incompletely recessive with a degree of dominance of -0.64. The Weak-R and Strong-R strains were then crossed. Analysis ofthe F1 and F2 progenies of this reciprocal cross revealed that the strong resistance phenotype was coded by a combination of the genes already present in the Weak-R genotype plus an extra major, incompletely recessive gene. There was also evidence of a minor dominant gene present in approximately 5% of Strong-R individuals.     

Gene interactions constrain the course of evolution of phosphine resistance in the lesser grain borer, Rhyzopertha dominica

Phosphine, a widely used fumigant for the protection of stored grain from insect pests, kills organisms indirectly by inducing oxidative stress. High levels of heritable resistance to phosphine in the insect pest of stored grain, Rhyzopertha dominica have been detected in Asia, Australia and South America. In order to understand the evolution of phosphine resistance and to isolate the responsible genes, we have undertaken genetic linkage analysis of fully sensitive (QRD14), moderately resistant (QRD369) and highly resistant (QRD569) strains of R. dominica collected in Australia. We previously determined that two loci, rph1 and rph2, confer high-level resistance on strain QRD569, which was collected in 1997. We have now confirmed that rph1 is responsible for the moderate resistance of strain QRD369, which was collected in 1990, and is shared with a highly resistant strain from the same geographical region, QRD569. In contrast, rph2 by itself confers only very weak resistance, either as a heterozygote or as a homozygote and was not discovered in the field until weak resistance (probably due to rph1) had become ubiquitous. Thus, high-level resistance against phosphine has evolved via stepwise acquisition of resistance alleles, first at rph1 and thereafter at rph2. The semi-dominance of rph2 together with the synergistic interaction between rph1 and rph2 would have led to rapid selection for homozygosity. A lack of visible fitness cost associated with alleles at either locus suggests that the resistance phenotype will persist in the field.     

Genetic linkage analysis of the lesser grain borer Rhyzopertha dominica identifies two loci that confer high-level resistance to the fumigant phosphine

High levels of inheritable resistance to phosphine in Rhyzopertha dominica have recently been detected in Australia and in an effort to isolate the genes responsible for resistance we have used random amplified DNA fingerprinting (RAF) to produce a genetic linkage map of R. dominica. The map consists of 94 dominant DNA markers with an average distance between markers of 4.6 cM and defines nine linkage groups with a total recombination distance of 390.1 cM. We have identified two loci that are responsible for high-level resistance. One provides approximately 50x resistance to phosphine while the other provides 12.5x resistance and in combination, the two genes act synergistically to provide a resistance level 250x greater than that of fully susceptible beetles. The haploid genome size has been determined to be 4.76 x 10(8) bp, resulting in an average physical distance of 1.2 Mbp per map unit. No recombination has been observed between either of the two resistance loci and their adjacent DNA markers in a population of 44 fully resistant F5 individuals, which indicates that the genes are likely to reside within 0.91 cM (1.1 Mbp) of the DNA markers.     

The rph1 gene is a common contributor to the evolution of phosphine resistance in independent field isolates of Rhyzopertha dominica

Phosphine is the only economically viable fumigant for routine control of insect pests of stored food products, but its continued use is now threatened by the world-wide emergence of high-level resistance in key pest species. Phosphine has a unique mode of action relative to well-characterised contact pesticides. Similarly, the selective pressures that lead to resistance against field sprays differ dramatically from those encountered during fumigation. The consequences of these differences have not been investigated adequately. We determine the genetic basis of phosphine resistance in Rhyzopertha dominica strains collected from New South Wales and South Australia and compare this with resistance in a previously characterised strain from Queensland. The resistance levels range from 225 and 100 times the baseline response of a sensitive reference strain. Moreover, molecular and phenotypic data indicate that high-level resistance was derived independently in each of the three widely separated geographical regions. Despite the independent origins, resistance was due to two interacting genes in each instance. Furthermore, complementation analysis reveals that all three strains contain an incompletely recessive resistance allele of the autosomal rph1 resistance gene. This is particularly noteworthy as a resistance allele at rph1 was previously proposed to be a necessary first step in the evolution of high-level resistance. Despite the capacity of phosphine to disrupt a wide range of enzymes and biological processes, it is remarkable that the initial step in the selection of resistance is so similar in isolated outbreaks.     

The rph2 gene is responsible for high level resistance to phosphine in independent field strains of Rhyzopertha dominica

The lesser grain borer Rhyzopertha dominica (F.) is one of the most destructive insect pests of stored grain. This pest has been controlled successfully by fumigation with phosphine for the last several decades, though strong resistance to phosphine in many countries has raised concern about the long term usefulness of this control method. Previous genetic analysis of strongly resistant (SR) R. dominica from three widely geographically dispersed regions of Australia, Queensland (SR(QLD)), New South Wales (SR(NSW)) and South Australia (SR(SA)), revealed a resistance allele in the rph1 gene in all three strains. The present study confirms that the rph1 gene contributes to resistance in a fourth strongly resistant strain, SR2(QLD), also from Queensland. The previously described rph2 gene, which interacts synergistically with rph1 gene, confers strong resistance on SR(QLD) and SR(NSW). We now provide strong circumstantial evidence that weak alleles of rph2, together with rph1, contribute to the strong resistance phenotypes of SR(SA) and SR2(QLD). To test the notion that rph1 and rph2 are solely responsible for the strong resistance phenotype of all resistant R. dominica, we created a strain derived by hybridising the four strongly resistant lines. Following repeated selection for survival at extreme rates of phosphine exposure, we found only slightly enhanced resistance. This suggests that a single sequence of genetic changes was responsible for the development of resistance in these insects.     

Control of Rhyzopertha dominica in stored rough rice through a combination of diatomaceous earth and varietal resistance

Adults ofRhyzopertha dominica （F.）, the lesser grain borer, were exposed on four varieties of rough rice with Dobie indices of susceptibility of 1.1 to 1.1 （low）, and four varieties with Dobie indices of susceptibility of 3.4 to 3.8 （high）. The varieties with low and high Dobie indices were classified as resistant and susceptible, respectively, to R. dominica. The purpose of the study was to evaluate control of R. dominica through the use of diatomaceous earth （DE） in combination with rice varieties that were either susceptible or resistant to R. dominica. The rice was treated with varying rates of the commercial DE Insecto, up to a maximum of 1 000 mg DE/kg of rice. Adult mortality at each application rate of DE was generally greater on three of four resistant varieties compared to three of four susceptible varieties. Progeny production from the parental generation exposed on the rice was also greater in 3 of the 4 resistant varieties compared to 3 of the 4 susceptible varieties at DE rates of 500 mg/kg or more. Progeny production in rice treated with a maximum rate of 1 000 mg/kg DE ranged from 7-44 adults on the resistant varieties compared to 75-155 adults on the susceptible varieties. At DE rates of 500, 750, and 1 000 mg/kg, the percentage of insect-damaged kernels （IDK） was also greater in 3/4 resistant varieties than in the susceptible varieties. Results show combining the use of DE with varietal resistance of rough rice to R. dominica could be used to limit populations of this insect in stored rice and help prevent economic damage.     

Pyrethroid-Acarophenax lacunatus interaction in suppressing the beetle Rhyzopertha dominica on stored wheat

Any biocontrol agent used against stored grain insects will have to interact with insecticides used as grain protectants due to their heavy use especially in tropical areas. Therefore, the interaction between the pyrethroid insecticides bifenthrin and deltamethrin and Acarophenax lacunatus (Cross & Krantz) (Prostigmata: Acarophenacidae), an egg parasite of the stored cereal pest Rhyzopertha dominica(F.) (Coleoptera: Bostrichidae), was assessed in a range of mite densities and insecticide doses for each compound. Despite the mite species presence on all insecticide doses, the lowest instantaneous rate of increase of A.lacunatus was recorded with the highest doses of insecticides. The presence of the biological control agent at all doses of both insecticides suggests its tolerance to these compounds. A. lacunatusshowed higher parasitism rate with bifenthrin than with deltamethrin indicating a higher selectivity of the first compound to the biocontrol agent. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification and purification of a distinct dihydrolipoamide dehydrogenase from pea chloroplasts

Two distinct dihydrolipoamide dehydrogenases (E3s, EC 1.8.1.4) have been detected in pea (Pisum sativum L. cv. Little Marvel) leaf extracts and purified to at or near homogeneity. The major enzyme, a homodimer with an apparent subunit M_r value 56 000 (80–90% of overall activity), corresponded to the mitochondrial isoform studied previously, as confirmed by electrospray mass spectrometry and N-terminal sequence analysis. The minor activity (10–20%), which also behaved as a homodimer, copurified with chloroplasts, and displayed a lower subunit M_r value of 52 000 which was close to the M_r value of 52 614±9.89 Da determined by electrospray mass spectrometry. The plastidic enzyme was also present at low levels in root extracts where it represented only 1–2% of total E3 activity. The specific activity of the chloroplast enzyme was three-to fourfold lower than its mitochondrial counterpart. In addition, it displayed a markedly higher affinity for NAD⁺ and was more sensitive to product inhibition by NADH. It exhibited no activity with NADP⁺ as cofactor nor was it inhibited by the presence of high concentrations of NADP⁺ or NADPH. Antibodies to the mitochondrial enzyme displayed little or no cross-reactivity with its plastidic counterpart and available amino acid sequence data were also suggestive of only limited sequence similarity between the two enzymes. In view of the dual location of the pyruvate dehydrogenase multienzyme complex (PDC) in plant mitochondria and chloroplasts, it is likely that the distinct chloroplastic E3 is an integral component of plastidic PDC, thus representing the first component of this complex to be isolated and characterised to date.Abbreviations E1 pyruvate dehydrogenase - E2 dihydrolipoamide acetyltransferase - E3 dihydrolipoamide dehydrogenase - PDC pyruvate dehydrogenase complex - OGDC 2-oxoglutarate dehydrogenase complex - GDC glycine decarboxylase complex - SDS-PAGE sodium dodecyl sulphate/polyacrylamide gel electrophoresis - TDP thiamine diphosphate - M_r relative molecular mass J.G.L. is grateful to the Biotechnology and Biological Sciences Research Council (BBSRC), U.K. for continuing financial support. M.C. is the holder of a BBSRC-funded earmarked Ph.D. studentship.     

玉米面水分含量对谷蠹生长发育和繁殖的影响

本文研究了玉米面的水分含量(21.33%、19.35%、18.67%、13.64%、10.98%)对谷蠹Rhyzopertha dominica Fabricius生长发育及产卵的影响。结果表明:玉米面的水分含量对谷蠹幼虫的发育历期影响显著,对卵和蛹的发育历期影响不显著;谷蠹卵的孵化率、幼虫和蛹的存活率在玉米面水分10.98%～19.35%的条件下增大,在玉米面水分21.33%条件下下降;谷蠹幼虫龄数也受到玉米面水分含量的影响,玉米面水分19.35%条件下幼虫龄数在3～5间变化,10.98%条件下幼虫龄数在3～8间变化;在试验的5种玉米面水分中,谷蠹只在19.35%和18.67%条件下才产卵,19.35%条件下日均产卵量和产卵期均大于18.67%条件下的相应值。     

Hydrogen bond network structures based on sulfonated phosphine ligands: The effects of complex geometry, cation substituents and phosphine oxidation on guanidinium sulfonate sheet formation

Interactions between guanidinium cations and the sulfonate groups on the phosphine [PPh₂C₆H₄-m-SO₃]⁻ have been exploited to incorporate iridium(I) centres into hydrogen-bonded networks. The crystal structure of [C(NH₂)₃]₂{trans-[IrCl(CO)(PPh₂C₆H₄-m-SO₃)₂]} (4) contains hexagonal guanidinium sulfonate (GS) sheets in which both of the sulfonate groups from each complex anion form hydrogen bonds within the same sheet. The crystal structures of [C(NH₂)₂(NHMe)][PPh₂C₆H₄-m-SO₃] (5) and [C(NH₂)₂(NHEt)][PPh₂C₆H₄-m-SO₃] (6) reveal that the GS sheets can tolerate the loss of one hydrogen bond donor, though twisting occurs to accommodate the alkyl group. However, the crystal structure of [C(NH₂)₂(NMe₂)][PPh₂C₆H₄-m-SO₃] (7) shows that ribbon structures are formed instead of sheets when two hydrogen bond donors are lost. The compound [C(NH₂)₂(NHMe)]₂{trans-[IrCl(CO)(PPh₂C₆H₄-m-SO₃)₂]} · 3/8H₂O (8) contains hydrogen-bonded cylinders as opposed to sheets. This is a likely consequence of a mismatch between the intramolecular S?S distance present in the anion, and the closer S?S distance present in a twisted GS sheet such as that in 5. The crystal structures of [C(NH₂)₂(NHEt)][P(O)Ph₂C₆H₄-m-SO₃] (9) and [C(NH₂)₂(NMe₂)][P(O)Ph₂C₆H₄-m-SO₃] · H₂O (10) show that the phosphine oxide group successfully competes with the sulfonate as a hydrogen bond acceptor. The crystal structure of 9 contains hydrogen-bonded ribbons that are interlinked through the anions which act as pillars to form a layer structure. In contrast, the crystal structure of 10 contains hydrogen-bonded sheets that involve cations, sulfonate groups, phosphine oxides and the included water molecule. These sheets are linked into a three-dimensional network through the anion pillars.     

A comparison of some effects of phosphine, hydrogen cyanide and anoxia in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae)

The effects of phosphine, hydrogen cyanide and anoxia on levels of ATP, pyruvate and lactate in Rhyzopertha dominica are compared. The effect of phosphine on anaerobiosis is not directly comparable either with HCN or anoxia. Reduction of catalase by feeding 3 amino 1,2,4 triazole does not enhance the toxicity of phosphine in treated insects.     

谷蠹感染的小麦储存环境中二氧化碳浓度变化研究

在25℃和密闭条件下测定了谷蠹Rhyzopertha dominica(F.)密度分别为0、2、5、10和20头/kg的小麦储存环境中二氧化碳浓度的变化情况。主要结果为:含水量为12%的小麦,未感染谷蠹成虫时,在180d内二氧化碳浓度从0.048%增加到1.157%;以2头/kg密度感染后,同样时间内二氧化碳浓度从0.048%上升到9.910%。害虫密度增加,产生的二氧化碳浓度都相应地增高,但二氧化碳的浓度与害虫密度不是相应地成比例升高。感染不同害虫密度的粮食,二氧化碳浓度随时间的延长呈"S"型曲线增长。这些结果表明,小麦感染谷蠹后在很短时间后即可检测到储存环境中二氧化碳浓度显著增加,二氧化碳浓度的变化与虫口密度相关。同样害虫密度时,储存时间延长,二氧化碳浓度呈"S"型曲线增加。一定条件下可通过检测二氧化碳浓度反映粮情和虫情变化。     

Population development of Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) on different stored products

The lesser grain borer, Rhyzopertha dominica, is an important pest of various stored products around the world. In this study, the development, survival, reproduction, and life table parameters of R. dominica were investigated on six stored products (angelica, jujube, maize, rice, soybean, and wheat). The developmental time of the immature stage of R. dominica was shortest on wheat (40.20 days) and longest on angelica (67.04 days). The survival rate of the immature stage was highest on wheat (76.33%) and lowest on angelica (41.00%). The fecundity level of R. dominica was highest on wheat (246.05 eggs/female) and lowest on angelica (69.38 eggs/female). The net reproductive rate (R₀) and intrinsic rate of increase (r_m) of R. dominica differed significantly among the six stored products. The highest R₀ of R. dominica was on wheat (68.50), followed by rice (41.28), maize (32.32), soybean (27.17), jujube (23.16), and angelica (20.18); the r_m values showed a similar trend, with values of 0.059, 0.046, 0.042, 0.039, 0.036, and 0.033, respectively. Our results indicate that wheat was the most suitable stored product, whereas angelica was the least suitable, for the feeding, development, and population increase of R. dominica. These findings provide basic information about the occurrence trends and characteristics of R. dominica that will be useful for the control of this pest on different stored products. The physicochemical properties of angelica should be further explored for potential application in the control or integrated management of R. dominica.     

RNA-Seq based transcriptome analysis reveals the molecular mechanism of triterpenoid biosynthesis in Glycyrrhiza glabra

Licorice is a frequently-used medicinal plant worldwide. Two triterpenoids, 18α-glycyrrhizic acid (18α-GC) and 18β-glycyrrhizic acid (18β-GC), are the key medicinal components accumulated in licorice. Biosynthesis of triterpenoids is a complex process that involves many secondary metabolic pathways. In this study, we tried to identify the key enzymes and pathways for the triterpenoid biosynthesis in licorice by analyzing the gene expression patterns in samples containing different GC levels. Glycyrrhizia glabra (one of the original species used as licorice in Chinese Pharmacopoeia) seeds were irradiated by X-ray and cultivated for one year, and samples with different GC contents were selected by HPLC analysis. RNA-Seq was performed to determine the gene expression in three X-ray irradiated G. glabra samples (H1, H2, and H3) with the highest GC content and one control G. glabra sample (L1) with the lowest GC content. 28.44 Gb raw data was generated and 47.7 million, 45.4 million, 43.3 million, and 45.9 million clean reads were obtained in samples H1, H2, H3, and L1, respectively. Approximately 48.53% of genes were annotated searching against GO and KEGG databases. A total of 1376 core differentially expressed genes (DEGs) were identified, which mainly enriched in phenylpropanoid metabolism, glycometabolism, plant circadian rhythm, and terpenoid biosynthetic pathway. 15 core DEGs selected from the 1376 DEGs were further verified by qRT-PCR, which confirmed that the RNA-Seq results were accurate and reliable. This study provides a basis for future functional genes mining and molecular regulatory mechanism elucidation of triterpenoid biosynthesis in licorice.     

Entomopathogenic fungus as a biological control agent against Rhyzopertha dominica F. (Coleoptera: Bostrychidae) on stored wheat

To evaluate the pathogenicity of Metarhizium anisopliae (Metschinkoff) Sorokin (Deuteromycotina: Hyphomycetes) a bioassay was designed under laboratory conditions against Rhyzopertha dominica F. (Coleoptera: Bostrychidae) on stored wheat. The fungus was applied at the dose rates of 8 × 10³, 8 × 10⁵, 8 × 10⁷ and 8 × 10⁹ conidia/kg of wheat and the bioassay was conducted at 25°C with 60% relative humidity. The data regarding the mortality was recorded after 7 and 14 days exposure intervals. All the treatments gave the significant mortality of R. dominica and M. anisopliae of 8 × 10⁹ conidia/kg was found to be the most effective after a 14-day exposure interval. There was greater production of progeny when the low rate of M. anisopliae was applied to wheat. Overall, our study showed that M. anisopliae is vigorous when applied at a high dose rate which revealed an effective control of R. dominica and also played a pivotal role in the integrated pest management program (IPM) of stored wheat insect pests.     

抗菌药物用量与耐药相关分析

目的研究抗菌药物用量与耐药的关系。方法对1998年至2002年我院住院病人抗菌药物的用量与医院感染主要病原菌的耐药进行相关分析。结果头孢噻肟的用量与大肠埃希菌对头孢噻肟的耐药率呈显著正相关(r=0.911,P=0.032);亚胺培南/西司他丁的用量与铜绿假单胞菌对亚胺培南的耐药率呈显著正相关(r=0.996,P=0.004)。结论加强抗菌药物的合理使用有助于改善当前微生物敏感性降低的形势。