Molecular Pathogens 2024, Vol.15, No.3, 129-141 http://microbescipublisher.com/index.php/mp 135 among treatment groups. Colonies exposed to the NPFM treatment had fewer workers and males, suggesting a detrimental effect of combined stressors. Panel (d) highlights the number of brood cells, with the control group having higher brood production compared to most treated groups, again emphasizing the negative impact of stressors on colony health. Lastly, panel (e) shows the number of queen cells produced, with significant differences observed across groups, indicating that stress exposure can affect reproductive output. Overall, the data suggest that exposure to multiple stressors severely impacts the health and reproductive success of bumblebee colonies. The combined effects of multiple stressors, including pathogens, pesticides, and environmental changes, pose a significant threat to honeybee populations. Understanding these interactions and their impacts at individual, colony, and population levels is crucial for developing effective conservation and management practices to ensure the sustainability of pollination services. 6 Monitoring and Diagnostic Techniques 6.1 Surveillance methods for pathogens Surveillance of pathogens in honeybee populations is crucial for understanding and mitigating the threats posed by diseases. Various methods have been developed to monitor the presence of pathogens such as Varroa mites and Nosema microsporidia. For instance, a study in Greece utilized LC-ESI-QqQ-MS and GC-EI-QqQ-MS methods to detect multiple active substances and metabolites in honeybee samples, which also included assessments for Varroa and Nosema infections. This comprehensive approach provided an integrated picture of the stressors impacting bee survival (Kasiotis et al., 2021). Such methodologies are essential for early detection and management of pathogen-related threats to honeybee colonies. 6.2 Detection of pesticide residues The detection of pesticide residues in honeybee environments is a critical aspect of monitoring their exposure to harmful chemicals. Several advanced analytical techniques have been developed for this purpose. For example, a study in Spain developed an ultrasound-assisted extraction procedure followed by dispersive solid-phase extraction (d-SPE) and LC-MS/MS to evaluate pesticide residue levels in honeybees and corbicular pollen (García-Valcárcel et al., 2019). Another study in Denmark employed APIStrip-based passive sampling, which uses Tenax sorbent to monitor pesticide residues in honeybee colonies without harming the bees. This method allowed for long-term monitoring and provided comprehensive data on pesticide contamination (Murcia-Morales et al., 2020; Murcia-Morales et al., 2021). Additionally, a multi-residue analysis using modified QuEChERS methods combined with GC-ToF and LC-MS/MS (Daniele et al., 2018) was developed to quantify 80 environmental contaminants in honeys, honeybees, and pollens, demonstrating high sensitivity and accuracy (Wiest et al., 2011; Xiao et al., 2021). 6.3 Environmental monitoring tools Environmental monitoring tools are essential for assessing the broader impact of environmental changes on honeybee populations (Căuia et al., 2020). Honeybee colonies themselves serve as effective bioindicators due to their extensive foraging activities. For instance, a study in Italy used bee-collected pollen to monitor pesticide contamination over three years, revealing widespread contamination by agricultural pesticides (Tosi et al., 2018). Similarly, a study in Brazil demonstrated the use of bee pollen as a bioindicator of environmental contamination by developing a GC-MS/MS analytical method for multiresidue determination of pesticides in pollen (Oliveira et al., 2016). These tools not only help in detecting contaminants but also in understanding the extent and impact of environmental changes on honeybee health. In summary, the integration of advanced analytical techniques and the use of honeybee colonies as bioindicators provide robust methods for monitoring pathogens, pesticide residues, and environmental changes. These tools are vital for the early detection and management of threats to honeybee populations, ensuring their health and sustainability.
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