Sodium Caseinate

To test how the dietary source of EAAs influenced the intake target of adult worker bumblebees, each bee was presented with a choice of two solutions: a 0.5 mol l⁻¹ sucrose solution and another solution that contained 0.5 mol l⁻¹ sucrose with protein (sodium caseinate, Sigma-Aldrich, C8654) or the 10 EAAs at equimolar concentrations (Table 5). The AAs used were: methionine, tryptophan, leucine, lysine, valine, arginine, isoleucine, phenylalanine, threonine and histidine (all from Sigma-Aldrich). These AAs are essential for many insect species and were identified as ‘essential’ for honeybees by de Groot (1953) . Both of the EAA sources were dissolved in a 0.5 mol l⁻¹ sucrose solution made with deionized water. Diets were made to specific protein to carbohydrate ratios (P:C), where the carbohydrate concentration remained constant (0.5 mol l⁻¹ sucrose) (Tables 4 and 5). The caseinate solutions were based on weight-to-weight proportions; the EAA solutions were based on the molar ratio of the EAAs-to-sucrose as in Paoli et al. (2014a (link),b (link)). Our diets did not have the same proportion of EAAs: upon acid hydrolysis (see below), caseinate was digested to a specific proportion of EAA and non-EAA that was dominated by isoleucine, phenylalanine, glutamic acid and tyrosine (Table 4). Furthermore, the most concentrated amino acids were in some cases three or four orders of magnitude higher than the least concentrated amino acids. In contrast, our EAA diet was nearly equimolar with a similar proportion w/w.
Table 5.

Ratios of dietary source of EAA:C

Diet tubes were weighed and replaced every 24 h. To adjust for evaporation, evaporation rates for each solution were measured in boxes containing the solutions (without bees). The average value for each solution was subtracted from the final weights for the consumption of each diet solution. Values for the amount of carbohydrate or protein and EAAs consumed were determined by dividing the weight of the consumed solution by its density (1.06) to obtain the volume. The amount of each solute in the solution was then obtained for the volume of solution consumed; this amount was combined to give a single value for consumption of protein and carbohydrate for each day. Total consumption was a measure of the total amount eaten over the 7 day period.

Indirect and sandwich enzyme immunoassays were performed as described before [20 (link),28 (link),32 (link),49 (link)].
For detection of rHbl components, established mAbs were used (1A12 and 8B12 [32 (link)] for Hbl L₂, 1E9 [29 (link)] for Hbl L₁ and 1B8 [29 (link)] for Hbl B), as well as the ones newly generated in this study (Table S1). To determine relative affinities of the newly generated mAbs, B. cereus culture supernatants and rHbl components were used in indirect EIAs. For that, dilution series of the antigens were applied, followed by the cell culture supernatants (constantly 1:20 in PBS). The relative affinity corresponds with the dilution that results in an absorbance value of 1.0. The productivity of the hybridoma cell lines was also determined in indirect EIAs. Supernatant of B. cereus MHI 1532 was applied (constantly 1:10 in PBS), followed by dilution series of the cell culture supernatants.
rHbl complex formation was investigated using indirect and sandwich EIAs. In the indirect assay, the microtiter plate was coated with a serial dilution of rHbl L₁ (120–0 pmol/mL) overnight. After washing, the second rHbl component was applied in constant concentration (60 pmol/mL) for 1 h. After blocking for 30 min with 3% sodium-caseinate in PBS, HblB-specific mAb 1B8 [29 (link)] and Hbl L₂-specific mAb 1H9 (this study) (2 μg/mL in PBS) were applied, respectively. After additional washing, rabbit-anti-mouse-HRP conjugate (1:2000 in 1% sodium-caseinate in PBS) was applied for detection. In the sandwich assay, the microtiter plate was coated with 10 μg/mL mAbs 1E9 [29 (link)] or 1G8 (this study), both Hbl L₁-specific, overnight. While the plate was blocked for 30 min with 3% sodium-caseinate in PBS, a mixture of rHbl components (L₁+B or L₁+L₂; 1.5 pmol/μL each, ratios 1:1, 5:1, 1:5, 10:1 or 1:10) was incubated at RT. The mixtures were applied to the microtiter plate as serial dilution from 75 to 0 pmol/mL. After washing, a specific mAb conjugate was applied for detection (1:2000 in 1% sodium-caseinate in PBS; 1B8-HRP [29 (link)] against Hbl B or 1H9-HRP (this study) against Hbl L₂). Analogously to the detection of the rHbl components, sandwich EIAs were used to detect Hbl complexes in the supernatant of B. cereus strain F837/76, which was applied to the microtiter plates as serial dilution. One-site-binding curves were applied to depict the absorption values compared to the sample dilutions.