Diallyl Trisulfide Causes Male Infertility with Oligoasthenoteratospermia in Sitotroga cerealella through the Ubiquitin-Proteasome Pathway.

Essential oils extracted from plant sources along with their biologically active components may have negative effects on insects. Diallyl trisulfide (DAT) is an active component of garlic essential oil, and it exhibits multi-targeted activity against many organisms. Previously we reported that DAT induces male infertility and leads to apyrene and eupyrene sperm dysfunction in Sitotroga cerealella. In this study, we conducted an analysis of testis-specific RNA-Seq data and identified 449 downregulated genes and 60 upregulated genes in the DAT group compared to the control group. The downregulated genes were significantly enriched in the ubiquitin-proteasome pathway. Furthermore, DAT caused a significant reduction in mRNA expression of proteasome regulatory subunit particles required for ATP-dependent degradation of ubiquitinated proteins as well as decreased the expression profile of proteasome core particles, including ß1, ß2, and ß5. Sperm physiological analysis showed that DAT decreased the chymotrypsin-like activity of the 20S proteasome and formed aggresomes in spermatozoa. Overall, our findings suggest that DAT impairs the testis proteasome, ultimately causing male infertility characterized by oligoasthenoteratospermia due to disruption in sperm proteasome assembly in S. cerealella.

Silencing the triacylglycerol lipase (TGL) gene decreases the number of apyrene sperm and inhibits oviposition in Sitotroga cerealella.

Triacylglycerol lipase (TGL) is an essential lipid metabolism enzyme that also plays a critical role in energy metabolism; however, how it regulates other life processes is unknown. To investigate the functional role of TGL in moth reproduction, males Sitotroga cerealella were used as a model. The TGL gene was cloned and analysed. The results showed that the open reading frame of TGL was 1968 bp long and contained three conserved regions. TGL gene expression was higher in the larval and early adult stages than in the pupal stage, with the highest levels observed in the fat body, testis and accessory glands during the early adult stage. Moreover, after TGL in male adults was silenced through RNAi, the protein content in male accessory glands remained unchanged, and the spermatophore transferred into females mated with TGL-silenced males became small and empty; meanwhile, the number of apyrene sperm in the spermatophore was significantly reduced due to the reduction of apyrene sperm in males, which eventually led to the significant reduction of egg-laying amount. All of the findings suggest that TGL regulates the amount of sperm in male moths as well as the morphology and quality of spermatophores transferred to females after mating with treated males, implying that TGL is critical for Sitotroga cerealella's reproductive process.

Effects of Diallyl Trisulfide, an Active Substance from Garlic Essential Oil, on Energy Metabolism in Male Moth Sitotroga cerealella (Olivier).

This study investigated the effects of diallyl trisulfide (DAT), an active substance from garlic essential oil, on the metabolism of the main energy substances of pre- and postmating males of Sitotroga cerealella. Males at 12 h postemergence were fumigated with DAT at a concentration (LC10 = 0.010 µL/L) in a glass jar for 7 h. The main energy metabolites from pre- and postmating males were determined, including protein, triglyceride, glycogen, total soluble sugar, trehalose, and trehalase. The contents of total protein and total soluble sugar and the trehalase activity of premating males were significantly increased following DAT treatment, whereas the contents of protein from the accessory gland, triglyceride, glycogen, and trehalose were significantly decreased after treatment. Additionally, after mating, the total protein and soluble sugar contents were significantly increased and the glycogen content was significantly decreased in the treatment group relative to the levels in controls, but there was no significant difference observed in triglyceride, accessory gland proteins, trehalose content, or trehalase activity between the treatment and control groups. Furthermore, the changes in the main energy substances between pre- and postmating in males after the DAT treatment (∆DAT) were smaller than those in the control group (∆CK). This result indicated that DAT can accelerate the rate of metabolism in males at LC10, leading to the accumulation of greater levels of total soluble sugar to support life activities and to the increased synthesis of proteins to resist an adverse environment.

Effect of Diallyl Trisulfide on the Reproductive Behavior of the Grain Moth, Sitotroga cerealella (Lepidoptera: Gelechiidae).

The Angoumois grain moth, Sitotroga cerealella (Olivier, 1789) (Lepidoptera: Gelechiidae), is primarily a pest of stored products, that feeds inside the grain as larvae inducing significant economic loss in various stored commodities. Our previous studies proved that garlic essential oil and its active substances inhibit oviposition in moths. To further explore the effect on reproductive behavior and accurately interpret the results in terms of effective control of the moth population, moths were treated with diallyl trisulfide (DATS), an active substance of garlic essential oil, at a dose of 0.015 µL/L in air (LC20, sub-lethal concentration). The results showed that fecundity and the proportion of viable eggs significantly decreased when the moths were treated with LC20 DATS. Furthermore, female circadian mating rhythms and calling periodicity changed significantly after treatment. Compared with controls, the peak in mating occurred approximately 1 h earlier on the first day after DATS treatment, while it was significantly later on days 2 and 3. Moreover, mating frequency declined in presence of DATS compared with the controls. The percentage of females engaging in calling behavior decreased significantly with time, to less than 50%, 2 days after treatment, while a high calling percentage (>80%) was recorded for control moths on all 4 days. In addition, DATS had an inhibitory effect on the mating duration of S. cerealella. Moreover, a significant reduction was observed in the amount of sex pheromones extracted 8 h and 9 h after treatment. Our findings suggested that DATS has the potential to manipulate the moth population at LC20 and would be an efficient alternative to synthetic insecticides for the control of pests having low toxicity to non-target organisms and ecosystems.