Sperms were placed in the viewing chamber and live video pictures were visualized using a setup comprising an Olympus BX40 microscope (Olympus Optical Co., Tokyo, Japan), a 10-times-negative-phase objective and a Basler digital camera (model acA1920-155 Basler AG, Vision Technologies, Ahrensburg, Germany). The tissues were punctured with a needle in a PBS drop. Upon completion of copulation, the individual females from each glass vial were separated and the spermatophore in bursa copulatrix were dissected under stereomicroscope. Briefly, control and DAT-fumigated male moths were mated with female partners in a separate glass vial. Total sperm motility of spermatophore in bursa copulatrix of female moths mated with DAT-treated males and control was determined with computer-assisted sperm-motility analyzer (CASA), Hamilton thorn CEROS II. Transmission Electron Microscopy of Testes Each experiment consisted of two groups with three replicates from control (CK) and treated (DAT) moths. After treatment, adult males were collected and processed for subsequent experiments accordingly. Adult male moths fumigated with DAT at a dose of 0.010 μL/L had normal mating behavior and successful insemination. Ten adult males were treated by fumigation with DAT at a dose of 0.010 μL/L in glass pots (1000 mL, 10 cm diameter × 13 cm height) for 7 h, as previously described. Pupa were inspected hourly for adult emergence, after which one-day-old adult males were collected for experiments. Virgin adult moths were obtained from individual grain containing pupae placed in small glass tubes capped with cotton cloths and held at the laboratory as described above. This strain, from Wuhan, Hubei province, China, was used for experimental tests with DAT (purity > 90%, Sigma-Aldrich, Steinheim, Switzerland). cerealella associated with wheat was maintained in a laboratory at 28 ☌ ± 1 ☌ and 75% ± 5% relative humidity, with a photoperiod of 14:10 h (light: dark). Overall, this study reported, for the first time, that DAT impairs energy metabolism, inhibits dimorphic spermatogenesis, and decreases sperm motility, while these abnormalities in sperm lead to adult-male infertility. Moreover, the total sperm motility was reduced, while the wave-propagation velocity, amplitude, frequency, and wavelength were significantly decreased compared with the controls. Additionally, the lipid homeostasis and ATP contents in the male adults were significantly decreased after treatment. Meanwhile, the ultrastructural analysis of the sperm showed that the DAT caused malformed and aberrant structures of mitochondrial derivatives of dimorphic sperm. The results indicated that the DAT significantly decreased the dimorphic sperm number. The effects on testis morphology, sperm number, motility, and lipid homeostasis were observed in adult males fumigated with DAT at a dose of 0.01 μL/L in air. The current study tested the capacity of DAT to decrease the male fertility of Sitotroga cerealella.

Diallyl trisulfide (DAT) is a biologically active component of garlic essential oil and exhibits multi-targeted activity against many organisms.

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