Abstract
Background:
Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) causes economic damage to stored foods, especially wheat, and is controlled by synthetic insecticides. Synthetic pesticides work but are expensive, non-biodegradable, pesticide-resistant, and harmful to the environment. Green-synthesized nanoparticles may improve pesticides. Traditionally, T. castaneum is identified by adult morphology. Pest management programs still struggle to identify this species quickly and accurately.

Aim:
Using species-specific PCR and real-time quantitative PCR with SYBR Green dye. The second purpose was to produce milk thistle seed AgNPs and test their bioinsecticide efficacy against T. castaneum.

Methods:
To identify T. castaneum, real-time qPCR was used with specific primers for the mt-COI gene fragment. Production and evaluation of silver nanoparticles synthesized from aqueous extract of Silybum marianum seed (SM-AgNPs) against the red flour beetle. The synthesized AgNPs were characterized. In the bioassay of biopesticides, SM-AgNPs were added to flour at 0.75, 1.5, 3, and 6 mg/g to evaluate their larvicidal (2nd and 5th larval instar) and adulticidal activities on T. castaneum. Mortality was reported at 24, 48, 72, and 96 hours following application.

Results:
PCR amplified a 330-bp COI gene fragment, and mtCOI gene qPCR identified T. castaneum efficiently, quickly, and species- specifically. The synthesized SM-AgNPs were characterized. FTIR spectra showed milk thistle functional groups. SEM showed circular to oval particles. The UV-Vis of SM-AgNPs showed significant absorption bands at 408 nm. AgNP particles averaged 119 nm and had a zeta potential of -48 mV. The bioassay results demonstrated significant dose- and time-dependent larvicidal and adulticidal effects of SM-AgNPs (p < 0.05). Second-instar larvae were most responsive to SM-AgNPs, while adults were least. At a concentration of 0.75 mg/g, SM-AgNPs showed the lowest mortality rate (5.6%) in adult red flour beetles after 24 h of exposure. In contrast, the highest mortality rate (70%) was observed in second-instar larvae after the fourth day of exposure to the highest concentration of SM-AgNPs (6 mg/g).

Conclusion:
SYBR Green real-time qPCR identified T. castaneum rapidly and accurately. The mortality rates at different nanoparticle concentrations and exposure times showed SM-AgNPs could be an eco-friendly and effective biological control approach for this pest species.

Key words: AgNPs; Bioinsecticide; Milk thistle; qPCR; Tribolium castaneum.