Role of Lipids in Phosphine Resistant Stored-Grain Insect Pests Tribolium castaneum and Rhyzopertha dominica.

Insects rely on lipids as an energy source to perform various activities, such as growth, flight, diapause, and metamorphosis. This study evaluated the role of lipids in phosphine resistance by stored-grain insects. Phosphine resistant and susceptible strains of the two main stored-grain insects, Tribolium castaneum and Rhyzopertha dominica, were analyzed using liquid chromatography-mass spectroscopy (LC-MS) to determine their lipid contents. Phosphine resistant strains of both species had a higher amount of lipids than susceptible stains. Significant variance ratios between the resistant and susceptible strains of T. castaneum were observed for glycerolipids (1.13- to 53.10-fold) and phospholipids (1.05- to 20.00-fold). Significant variance ratios between the resistant and susceptible strains of R. dominica for glycerolipids were 1.04- to 31.50-fold and for phospholipids were 1.04- to 10.10-fold. Glycerolipids are reservoirs to face the long-term energy shortage. Phospholipids act as a barrier to isolate the cells from the surrounding environment and allow each cell to perform its specific function. Thus, lipids offer a consistent energy source for the resistant insect to survive under the stress of phosphine fumigation and provide a suitable environment to protect the mitochondria from phosphine. Hence, it was proposed through this study that the lipid content of phosphine-resistant and phosphine-susceptible strains of T. castaneum and R. dominica could play an important role in the resistance of phosphine.

Preliminary Study on the Differences in Hydrocarbons Between Phosphine-Susceptible and -Resistant Strains of Rhyzopertha dominica (Fabricius) and Tribolium castaneum (Herbst) Using Direct Immersion Solid-Phase Microextraction Coupled with GC-MS.

Phosphine resistance is a worldwide issue threatening the grain industry. The cuticles of insects are covered with a layer of lipids, which protect insect bodies from the harmful effects of pesticides. The main components of the cuticular lipids are hydrocarbon compounds. In this research, phosphine-resistant and -susceptible strains of two main stored-grain insects, T. castaneum and R. dominica, were tested to determine the possible role of their cuticular hydrocarbons in phosphine resistance. Direct immersion solid-phase microextraction followed by gas chromatography-mass spectrometry (GC-MS) was applied to extract and analyze the cuticular hydrocarbons. The results showed significant differences between the resistant and susceptible strains regarding the cuticular hydrocarbons that were investigated. The resistant insects of both species contained higher amounts than the susceptible insects for the majority of the hydrocarbons, sixteen from cuticular extraction and nineteen from the homogenized body extraction for T. castaneum and eighteen from cuticular extraction and twenty-one from the homogenized body extraction for R. dominica. 3-methylnonacosane and 2-methylheptacosane had the highest significant difference between the susceptible and resistant strains of T. castaneum from the cuticle and the homogenized body, respectively. Unknown5 from the cuticle and 3-methylhentriacontane from the homogenized body recorded the highest significant differences in R. dominica. The higher hydrocarbon content is a key factor in eliminating phosphine from entering resistant insect bodies, acting as a barrier between insects and the surrounding phosphine environment.

New Method of Analysis of Lipids in Tribolium castaneum (Herbst) and Rhyzopertha dominica (Fabricius) Insects by Direct Immersion Solid-Phase Microextraction (DI-SPME) Coupled with GC-MS.

Lipids play an essential role in providing energy and other physiological functions for insects. Therefore, it is important to determine the composition of insect lipids from cuticular and internal tissues for a better understanding of insect biology and physiology. A novel non-derivatization method for the analysis of lipids including fatty acids, hydrocarbon waxes, sterols in Tribolium castaneum (Herbst) and Rhyzopertha dominica (Fabricius) was explored using the direct immersion solid-phase microextraction (DI-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). Nine extraction solvents, acetonitrile, methanol, hexane, ethanol, chloroform, acetonitrile and ethanol (1:1 v/v), acetonitrile and water (1:1 v/v), ethanol and water (1:1 v/v) and acetonitrile and ethanol and water (2:2:1 v/v/v) were selected and evaluated for the extraction of insect lipids with DI-SPME fiber. Acetonitrile extraction offered the best qualitative, quantitative, and number of lipids extracted from insects samples results. Acetonitrile extracted high-boiling point compounds from both species of tested insects. The range of hydrocarbons was C25 (pentacosane) to C32 (dotriacontane) for T. castaneum and C26 (11-methylpentacosane) to C34 (tetratriacontane) for R. dominica. The major compounds extracted from the cuticular surface of T. castaneum were 11-methylheptacosane (20.71%) and 3-methylheptacosane (12.37%), and from R. dominica were 10-methyldotriacontane (14.0%), and 15-methyltritriacontane (9.93%). The limit of detection (LOD) for the n-alkane compounds ranged between 0.08 (nonacosane) and 0.26 (dotriacontane) µg/g and for the fatty acids between 0.65 (arachidic acid) to 0.89 (oleic acid) µg/g. The study indicated that DI-SPME GC-MS is a highly efficient extraction and a sensitive analytical method for the determination of non-derivatized insect lipids in cuticular and homogenized body tissues.

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 ozone on respiration of adult Sitophilus oryzae (L.), Tribolium castaneum (Herbst) and Rhyzopertha dominica (F.).

The effect of ozone on the respiration of three species of adult stored-product Coleoptera was tested in an air-tight flask. Sitophilus oryzae (L.), Rhyzopertha dominica (F.) and Tribolium castaneum (Herbst) adults were exposed to atmosphere containing 0.1, 0.2 or 0.4microg/ml initial ozone at 23-25 degrees C and 50% r.h. Carbon dioxide (CO(2)) production reflected the respiration rates of insects and was determined with a gas chromatograph (GC). The experiments showed that the effects of ozone on respiration had two distinct phases. Phase 1 involved a lower respiration rate of the adult stored-product Coleoptera under ozone atmosphere and reflected the need for insects to reduce ozone toxicity. After 1h, CO(2) production of S. oryzae was 3.19, 2.63, 2.27 and 1.99microl/mg for the ozone concentration of 0, 0.1, 0.2 and 0.4microg/ml, respectively. The results also showed that there were decreases in the rate of respiration in R. dominica and T. castaneum with an increase in ozone concentration. During phase 2, respiration of S. oryzae, R. dominica, and T. castaneum adults treated with ozone increased as the ozone degraded to oxygen. After 7h, the effect of ozone on CO(2) production, relative to the control, changed from a decrease to an increase. The findings in relation to control strategies were discussed.

Proteomic evaluation of adults of Rhyzopertha dominica resistant to phosphine.

A proteomic study using a PH(3)-susceptible (RD2) and -resistant strain (CRD343) of Rhyzopertha dominica was undertaken to validate the relation between change of proteins and PH(3) resistance. Protein expression levels were compared using PD-Quest program after two-dimensional polyacrylamide gel-electrophoresis. Comparing the intensity of proteins, 15 proteins decreased and 6 proteins were newly expressed in CRD343. After MALDI-TOF and LC-MS/MS analyses, the decreased proteins were identified as arginine kinases, dihydrolipoamide dehydrogenase, Hsp60, reverse transcriptase, glyceraldehydes-3-phosphate dehydrogenase, triosephosphate isomerase and hypothetical proteins. Dihydrolipoamide dehydrogenase is involved in the Krebs cycle and glyceraldehydes-3-phosphate dehydrogenase and triosephosphate isomerase are involved in the glycolysis pathway. Among up-regulated proteins, sodium channel, glutamate racemase, enolase and vitellogenin were identified. Taken together, PH(3) affected glycolysis as well as Krebs cycle and the induction of enolase might recover this dysfunction.