Optimization Method for Proteomic Analysis of the Larva and Adult Tissues of Plutella xylostella (L.) (Lepidoptera: Plutellidae)

Abstract

Plutella xylostella (L.) (Lepidoptera: Plutellidae), the major insect pest of cruciferous crops worldwide shows significant resistance to almost all classes of insecticides. In order to effectively prevent and manage the insecticidal resistance, it is crucial to understand the physiological adaptation of insects against insecticides. Identification of insect protein that interacting with insecticides and characterization of their modification in resistant strains can be done by using differential proteomics approach. This study focuses on optimizing a sensitive and rapid method for the extraction of high quality protein of both larva and adult tissues of P. xylostella to be used in two-dimensional gel electrophoresis. Five extraction methods were evaluated for protein concentration, yields and resolving patterns of one-dimensional and two-dimensional electrophoresis. The results showed that trichloroacetic acid/acetone extraction methods with two different concentrations of 2-mercaptoethanol produced the highest protein concentration and yield for both adult and larva tissues, respectively. Meanwhile, trichloroacetic acid/acetone with dithiothreitol extraction method gave better separation of spots and intensity for both larva and adult tissues compared to other methods tested. As such, we concluded that trichloroacetic acid/acetone with dithiothreitol successfully yielded high total protein concentration and good separation of two-dimensional electrophoresis gel spots in both adult and larva P. xylostella.

Toxicity and Sublethal Effect of Farnesyl Acetate on Diamondback Moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae)

“…The mortality of the larvae was observed and recorded after 96 h exposure to the treatments. The insects were considered dead when they were unable to move after being prodded with a soft paint brush [ 37 ].…”

Evaluation of femaleAedes aegyptiproteome via LC-ESI-MS/MS using two protein extraction methods

“…The TCA/acetone base extraction protocol is one of the most reported protein extraction methods from various samples, including insects. TCA acetone protein extraction method has continued to be an effective method in reducing protein degradation and get rid of interfering elements (Hassan et al, 2018). Visual observation of distinctive protein bands with no smearing showed good quality of protein extract accomplished using both methods in this study (Fig.…”

“…These included TCA acetone with 0.7% 2-mercaptoethanol, TCA acetone with 2% 2-mercaptoethanol, TCA acetone with 40 mM DTT, lysis buffer (7M urea, 2M thiourea, 4% CHAPS) Tris HCL method and PBS method. They concluded that TCA acetone with 40 mM DTT yielded high total protein amount of 25.17 mg/g with the most abundant protein spots of about 683 (Hassan et al, 2018). The TCA/acetone base extraction protocol is one of the most reported protein extraction methods from various samples, including insects.…”

Characterization of Antibacterial Peptides in Culex pipiens (Diptera: Culicidae) in Response to Bacillus sphaericus infection

“…Protein extraction was performed as follow in which samples were homogenized and extracted using a buffer of Tris and NaCl at pH 8.0. The homogenates were spinned for 10 minutes at 10,000 Xg and then supernatants were collected (HASSAN et al (2018).…”