Differential Proteome Analysis Of The Male And Female Antennae From Holotrichia Parallels Motschulsky

The scarab beetle, Holotrichia parallela Motschulsky is harmful on plants andposes a worldwide agricultural problem. New interventions are required to optimallyand sustainably control the scarab beetle. The scarab beetle olfactory system isimportant in host seeking and mate finding. Understanding olfactory function couldlead to development of control strategies in the field, such as retrofit the fatal trap thatrepelling or attracting insect pests. Comparative antenna proteomics are a relativelyuseful technique that can be used to investigate olfaction related protein alterationsthat can help in understanding olfaction recognition mechanism for antenna. Tounderstand the olfactory mechanism of H. parallela antennae in detecting specificvolatile compound in the environment, proteins profiles of H. parallela female versusmale antenna were analyzed using two-dimensional electrophoresis (2-DE) followedby a mass spectrometry and bioinformatics analysis.Protein sample preparation is a crucial step in a2-DE proteomics approach. Inorder to establish a routine protocol for the application of proteomics analysis toantenna, this study focuses on the specific protein extraction problems in insect tissuesand evaluates four methods to bypass them. The approaches of TCA/acetoneprecipitation, phenol extraction methanol/ammonium acetate precipitation (PA), PEGprecipitation, and no precipitation were evaluated for proteins isolation andpurification from adult antenna for H. parallela. Analysis of protein yield, imagequality and spot numbers demonstrate that the TCA/acetone precipitation protocol is areproducible and reliable method for extracting proteins from antenna.Comparative proteomic analysis was used to investigate the abundance changesof protein profiles from antennae of H. parallela females and males. Approximately1100protein spots in silver stained were reproducibly and repeatedly detectedbetween gels by the Image Master2D Platinum software. Quantitative image analysisrevealed a total of47protein spots that showed significant and reproducible changes(>1.5fold and p-value <0.05)) in female versus male adult antennae. Twenty five ofthese protein spots were significant abundant in male antennae than female, while22protein spots were more abundant in female antennae. To identify the differentially expressed proteins in antenna, the protein spots were excised from the preparative gelsand analyzed using MALDI-TOF/TOF MS. Thus,35of the47differentiallyexpressed proteins were positively identified. Among these,65.7%are proteins thatinvolved in carbohydrate and energy metabolism, antioxidant system, transport, andamino acid/nucleotide metabolism. Many proteins identified here were not previouslyreported in olfaction recognition. These proteins include peptidoglycan recognitionprotein-1（spot16）, acyl-CoA dehydrogenase (spot26), similar to triosephosphateisomerase (spot29), aldo-keto reductase-like (spot34)、serine protease (spot21),odorant binding protein2(spot38), aldehyde dehydrogenase (spot14), antennae-richcytochrome P450（spot12）etc.These proteins were classified into8groups based on the biological function ofKEGG database and Gene Ontology database. The majority of the protein wascarbohydrate and energy metabolism related proteins (20.0%), followed byantioxidant system proteins (17.1%), transport proteins (14.3%), and the amino acidand nucleotide metabolism proteins (14.3%). In addition, cytoskeleton proteins(11.4%), and protein folding function (8.6%) also took up a great part of identifiedproteins.Hence, from the identified proteins, a total of6proteins were recognized byinteractions between the imported proteins that showed probable biological relevancein the context of olfaction recognition for antenna from the existing evidence arehighlighted. Among these proteins, six important olfaction related proteins includingheat shock70kDa protein cognate3, serine protease, aldo-keto reductase-like wereup-regulated in males, whereas antennae-rich cytochrome P450, aldehydedehydrogenase, putative glutamine synthetase were up-regulated in females forantennae. Alterations in the levels of these proteins were further confirmed byRT-PCR.These results indicated that odorant response mechanisms are sex-specificbecause of natural selection for different olfactory functions and comparative proteinprofiles of female and male antennae are valuable sources for the identification of newplayers in chemical communication.