## 3 Results and Discussion
### MALDI-TOF/TOF Analysis and Protein Identification  
Differentially expressed protein spots were manually excised from the gels and digested with trypsin, and then analyzed by MALDI-TOF/TOF combined with a database search. Fifteen protein spots were successfully identified, and 24 protein spots could not be detected (Table 1). The identified proteins showed homology to hypothetical proteins from different organisms; however, the observed and theoretical data on pI or molecular weight (MW) were not always in agreement. These differences could be attributed to post-translational modifications, multiprotein isoforms, and different compositions between the _S. oryzae_ proteins and homologous known proteins. PH3 toxicity has been focused on the mitochondrial respiration by interaction with cytochrome c oxidase to cause depletion of ATP production [29]. Cytochrome oxidase subunit I (42.5 kDa, pI 7.33), cytochrome P450 (75 kDa, pI 4.96), heat shock protein 70 (HSP70, 66.1 kDa, pI 4.74), triosephosphate isomerase (21.7 kDa, pI 7.95), citrate synthase 2 (37.6 kDa, pI 7.99), delta-1-pyrroline-5-carboxylate dehydrogenase (55.0 kDa, pI 8.07), ATP synthase subunit beta (51.6 kDa, pI 7.21), and glyceraldehyde-3-phosphate dehydrogenase (28.1 kDa, pI 7.36) were overexpressed in the PH3-R strain compared with their expression in the PH3-S strain of _S. oryzae_. Figure 1: Representative 2-DE maps of proteins extracted from the PH3-S (**a**) and PH3-R (**b**) strains of _Stophilus oryzae_. The spots were visualized by silver staining. Differentially accumulated protein spots are indicated by green sashes and numbers. The 2-DE was repeated at least three times for each group. Cytochrome oxidase is targeted by phosphine [30; 31]. In the present study, the protein expression of cytochrome oxidase was markedly higher in the PH3-R strain (18.8-fold) than in the PH3-S strain of _S. oryzae_. The expression of excess cytochrome oxidase in the PH3-R strain compared with that in the PH3-S strain suggests that the PH3-R strain can tolerate the inhibitory effects of phosphine absorbed from functional doses. The upregulation of cytochrome P450 and HSP70 may lead to the detoxification of fumigants [32]. Additionally, HSP70 acts as a molecular sensor of oxidative stress. Phosphine induces oxidative stress in insects, mammalian cells, animals, and humans [33]. The primary mechanism of PH3 resistance in stored-grain insects is to absorb very little PH3 compared to their susceptible strains and an involves additional detoxification process of the fumigant [34]. Triosephosphate isomerase in the glycolysis pathway was upregulated in the PH3-R strain of _S. oryzae_. Additionally, citrate synthase 2, delta-1-pyrroline-5-carboxylate dehydrogenase, ATP synthase subunit beta, and glyceraldehyde-3-phosphate dehydrogenase in the mitochondrial respiratory chain were upregulated in the PH3-R strain compared with their expression in the PH3-S strain of _S. oryzae_. Similar results were found in [PERSON] et al. report where heat shock protein and ATP synthase subunit beta were overexpressed in _R. dominica_ exposed to MA [17]. The accumulation of the protons in the mitochondrial inner space drives the ATP synthase to synthesize ATP molecules from ADP and Pi. However, if O2 intake decreases, the ATP synthase catalyzes the reverse reaction working now as an ATPase [35]. However, [PERSON] et al. reported different results from our study. Glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase were dramatically down-regulated in the PH3-resistant strain of _R. dominica_[21]. In addition, beta-glucuronidase (144.1 kDa, pl 5.19), protein MLP1-like (37.2 kDa, pl 4.8), glycogen phosphorylase (90.4 kDa, pl 4.74), DNA primase large subunit (39.1 kDa, pl 4.96), and beta-actin (66.8 kDa, pl 4.5) were down-regulated in the PH3-R strain of _S. oryzae_. In summary, we only speculate on the involvement of these regulated genes in phosphine resistance because no phenotypic evidence is directly linked to these genes.