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This protocol was developed for the COST-Action “Super-B”, whose purpose was to coordinate research, outreach and policy towards sustainable pollination1-3. The protocol addresses the detection of parasites and pathogens across bee species, as one of several possible drivers of bee decline4,5. It consists of four major components:1. A sample collecting protocol, based around a dominant bee species driving pathogen distribution among other bee species2. A sample processing protocol, based on analyzing nucleic acids from individual bees3. A pathogen detection protocol, based on RT-qPCR with broad-range primers for several common pathogens4. A barcoding protocol, for accurate bee species identification The protocols have largely been adapted from existing knowledge and protocols but also include two key innovations: the use of passive reference nucleic acids and synthetic positive controls, that significantly improve the quality and robustness of the raw data, and thus the reliability of the analyses and conclusions.

Objective Locked-in syndrome (LIS) is a state of complete paralysis, except for ocular movements, which results from ventral brainstem lesions. Patients typically are fully conscious. Here we tested the hypothesis that electroencephalographic (EEG) rhythms are abnormal in LIS patients, possibly due to an impaired neural synchronization between brainstem and cerebral cortex. Methods Resting state eyes-closed EEG data were recorded in 13 LIS subjects and 15 cognitively normal control subjects. With reference to the individual alpha frequency (IAF), the bands of interest were delta (IAF-8 to IAF-6 Hz), theta (IAF-6 to IAF-4 Hz), alpha 1 (IAF-4 to IAF-2 Hz), alpha 2 (IAF-2 to IAF Hz), and alpha 3 (IAF to IAF + 2 Hz). Furthermore, beta 1 (13–20 Hz) and beta 2 (20–30 Hz) bands were also considered. Cortical EEG sources were estimated by low-resolution electromagnetic tomography (LORETA). Results The power of alpha 2 and alpha 3 sources in all regions was lower in patients with LIS compared to controls. The power of delta sources in central, parietal, occipital and temporal regions was higher in patients with LIS compared to controls. Conclusions These results suggest that cortical sources of resting state eyes-closed alpha and delta rhythms are abnormal in LIS patients. Significance LIS is accompanied by a functional impairment of cortical neuronal synchronization mechanisms in the resting state condition.