Vitellogenin appears in hemolymph at an earlier pupal stage in queens and at a higher concentration than in workers
Using rocket immunoelectrophoreses we corroborated previous data showing that vitellogenin appears earlier in queens than in workers. Queen hemolymph samples presented a small peak of immunoreactive vitellogenin in hemolymph from brown-eyed pupae with dark pigmented cuticle (Pbd), increased slightly in the next stage (pharate adult; Pha), and reached elevated levels in newly emerged and 1 to 3 day old bees (). Until the 3rd day after eclosion, worker hemolymph exhibited only low reaction peaks, compared to those of the queen (). These data show that vitellogenin levels in queens, are 10 times higher than that observed in workers during the first days of adult life.
Figure 1. Vitellogenin titers in hemolymph of pupal stages and young adults of Apis mellifera queens (A) and workers (B). Rocket immunoelectrophoreses with 3µg hemolymph proteins per well were carried out in 1% agarose gels containing 1% (more ...)
Utilizing Western blots as a more sensitive assay we detected vitellogenin in queen hemolymph at the Pbm stage, approximately 60h before eclosion (). In worker hemolymph vitellogenin was detected later at the Pha stage, about10h before eclosion (). It was occasionally possible to detect vitellogenin in the worker in an earlier developmental stage ().
Figure 2. Western blot analysis to determine the onset of vitellogenin synthesis in honeybee queen and worker pupae. SDS-PAGE (7.5%) was performed with 1µg of hemolymph proteins. After blotting to a PVDF membrane vitellogenin was detected by a rabbit (more ...)
Figure 5. Western blot analysis to determine the onset of vitellogenin synthesis in honeybee queen and worker pupae, showing the effect of ecdysone on vitellogenin expression. A) workers, and B) queens treated in Pbl stage with 5 µg of ecdysone (in ethanol/Ringer's (more ...)
Juvenile hormone advances vitellogenin appearance in both castes
Exogenous juvenile hormone applied to pink- and brown-eyed pupae had a pronounced effect on vitellogenin levels in both castes, advancing the onset of hemolymph vitellogenin appearance to earlier pupal stages. Western blot analyses indicated that in queen pupae treated with juvenile hormone, vitellogenin is expressed in PbmI, with increasing levels by Pbd, while in control pupae vitellogenin appeared only in PbmII, and remained at low levels (). A similar effect was observed in worker pupae treated with juvenile hormone, which responded with an earlier appearance of vitellogenin in the Pbm II stage (). Detectable vitellogenin levels were not seen in control worker pupal hemolymph during corresponding stages (). Levels of total hemolymph protein concentration () were not affected by the juvenile hormone treatments (Mann-Whitney test >0.05).
Figure 3. Western blot analysis to determine the onset of vitellogenin synthesis in honeybee queen and worker pupae. SDS-PAGE (7,5%) was performed with 1µg of hemolymph proteins. After blotting to a PVDF membrane vitellogenin was detected by a rabbit (more ...)
Figure 4. Protein concentrations (means SEM) in the hemolymph of A) queen and B) worker pupae of Apis mellifera treated with either 10 µg of juvenile hormone (in 2 µl acetone), or with 2 µl of acetone (solvent control) during the pink-eyed (more ...)
Juvenile hormone stimulates transcription of the vitellogenin gene
Juvenile hormone could either induce vitellogenin gene transcription, or trigger translation from a previously transcribed vitellogenin mRNA. To distinguish between these two possibilities, worker pupae received a single injection of 10µg of actinomycin D, which inhibits gene transcription, 1h before the topical application of 10µg of juvenile hormone-III. Hemolymph from Pbm workers treated with actinomycin and juvenile hormone was vitellogenin free, as judged by the Western blot assays, while hemolymph of control pupae, which received juvenile hormone but no actinomycin, had this protein in the Pbm III stage as expected (). These results suggest that juvenile hormone regulates vitellogenin synthesis primarily at the transcriptional level during the pupal stages.
High levels of ecdysteroids inhibit the appearance of vitellogenin synthesis in honeybee pupal hemolymph
Injection of 5 µg of ecdysone at the Pbl stage, delayed the appearance of vitellogenin in queen and worker pupae. Ecdysone-treated worker pupae did not exhibit detectable vitellogenin levels at an advanced Pbd stage, when this protein can occasionally be detected in controls (). Similarly, in ecdysone-treated queens, vitellogenin became apparent only in the pharate adult stage (Pha), representing almost a 48h delay as compared to its normal onset in the hemolymph (). Total protein levels were significantly higher in the hemolymph of ecdysone-treated worker pupae than in control worker pupae (Mann-Whitney test <0.05; ).