Percorrer por autor "Bejaoui, Mohamed Khalil"
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- Development of a Molecular Tool to Detect Mutations Associated with Varroa destructor Resistance to PyrethroidsPublication . Bejaoui, Mohamed Khalil; Henriques, Dora; Costa, Maíra; Ben Souf, IkramThe Western honey bee (Apis mellifera) is essential for agriculture and biodiversity, yet colonies are increasingly threatened by Varroa destructor, a parasitic mite that weakens hosts and vectors viruses such as Deformed Wing Virus (DWV). Control still relies heavily on acaricides, pyrethroids (fluvalinate, flumethrin), organophosphates (coumaphos) and formamidines (amitraz), but prolonged use has driven resistance. In V. destructor, pyrethroid resistance is strongly associated with mutations in the voltage-gated sodium channel (VGSC), particularly the M918L and L925V substitutions, which have been reported across Europe and motivate targeted surveillance in Portugal. This study aimed to develop a rapid LAMP (Loop-Mediated Isothermal Amplification) assay to detect VGSC resistance-associated mutations. A Portuguese baseline was first established by sequencing the VGSC Domain II fragment from 100 mites collected across 35 apiaries. Sanger genotyping showed that 38% of successfully analyzed mites carried the M918L/L925V resistant haplotype, 54% were wild type, and 8% remained unresolved after repeat testing, indicating marked geographic heterogeneity. Guided by curated alignments, two LAMP primer sets targeting the resistance region were designed (NEB LAMP Designer; LAMP DESIGNER v1.16), screened in silico for structural/thermodynamic suitability, and experimentally optimized using real-time fluorescence, with electrophoretic confirmation. Initial optimization on a CFX96 platform (63–67 °C) yielded amplification in both samples and no-template controls, with characteristic ladder-like products in NTCs, consistente with template-independent amplification driven by primer–primer and/or intra-primer interactions. Under more stringent conditions on QuantStudio™ 5 (68–70 °C), one primer set (NEB Set ID 36) achieved partial genotype discrimination at 69 °C, with earlier amplification in resistant than in wild-type samples and only late low-level signal in controls; the second primer set did not provide diagnostically useful separation. Overall, the work supports the potential of LAMP for rapid resistance screening, while highlighting that SNP genotyping demands stringent specificity and will require further primer refinement and validation prior to robust field implementation.
- Development of LAMP Primers for the Detection of Pyrethroid Resistance Mutations in Varroa destructorPublication . Costa, Maíra; Yadró Garcia, Carlos A.; Lopes, Ana Rita; Bejaoui, Mohamed Khalil; Almeida, Jhennifer; Correia, Lucas; Sánchez, Sara; Li, Fernanda; Pinto, M. Alice; Henriques, DoraVarroa destructor is one of the main threats to Apis mellifera L., directly affecting colony health and contributing to their global decline. Control of this mite is traditionally achieved using acaricides, with pyrethroids such as tau-fuvalinate and fumethrin being the most used, acting on voltage-gated sodium channels (VGSC). However, the intensive use of these compounds by beekeepers has led to the emergence of resistance, associated with mutations at residues 918 and 925 of the VGSC gene [1]. Traditional methods for detecting these mutations, such as PCR, TaqMan and RT-PCR, are effective but require expensive laboratory equipment. In this context, Loop-mediated Isothermal Amplification (LAMP) is a promising alternative, offering rapid and cost-effective detection without the need for thermal cycling [2]. LAMP is based on the use of a set of four to six primers, including two inner primers (FIP and BIP), two outer primers (F3 and B3), and optionally two loop primers (LoopF and LoopB), which are introduced to accelerate the amplification reaction [3]. This study aimed to develop specific LAMP primers, using the NEB LAMP software, for the detection of the main mutations associated with Varroa destructor resistance to pyrethroids in Portugal. The predictive efficiency, specificity, and thermodynamic properties of the designed primers were assessed using BLAST, eLAMP, and OligoAnalyzer tools, considering qPCR parameters. This work successfully identified specific primer sets, including loop primers, for the detection of the mutation at position 925, which may be used in future experimental validations for rapid diagnostic applications.