I work on hypopharyngeal glands of honeybee. How could I Measure and classify the hpg of honeybee?
If you are looking to analyze the composition of what the glands contain in great detail, I suggest collecting some gland extract and making fractions, firstly by centrifuging the extract to precipitate proteins, then making further fractions of the smaller metabolites based on polarity. I would suggest analyzing the proteins using MALDI-ToF-MS, and using LC-MS to analyze the non-volatile metabolites (reverse phase LC for non-polar metabolite analysis and HILIC LC for polar metabolites), operating in both positive and negative mode to obtain maximal metabolite ionization yield. If you have the luxury of a UPLC-MS, then your metabolite profiles will be much richer. To analyze the most volatile components, I would suggest solid phase micro-extraction (SPME) and subsequent GC-MS. If you're interested in identifying correct chirality of molecules, then I suggest using cyclodextrin based columns. If you're looking for differences in gland extracts between certain bee treatments, then a principal component analysis will cluster the samples, and if you have a split in clusters between treatments, then you know the metabolite profiles vary, and you could look at the loading plot to identify the metabolites which contributed most to the variation, and hence are (usually) accountable for the differences. Hope this helps! Feel free to message me if I've been a bit too complicated.
I was measuring the size of hpg and using this as parameter. The first I dissected the left and right HPG and next I calculated the size of HPG as the average size of 20 acini - the squere root of longest and shortest diameter of 10 right gland and 10 left gland acini. It is good discribed in some paper e.g. (Reversion in honeybee,< i> Apis mellifera, workers with different life expectancies K Kuszewska, M Woyciechowski - Animal Behaviour, 2013; Swarming generates rebel workers in honeybees, M Woyciechowski, K Kuszewska; Current Biology 22 (8), 707-711). The size of HPG is a good parametr describing a activation of these glands - (Deseyn, J., and Billen, J. (2005). Age-dependent morphology and ultrastructure of the hypopharyngeal gland of Apis mellifera workers (Hymenoptera, Apidae). Apidologie (Celle) 36 , 49–57; Amdam, G.V., Aase, A.L.T.O., Seehuus, S.C., Kim Fondrk, M., Norberg, K., and Hartfelder, K. (2005). Social reversal of immunosenescence in
hony bee workers. Exp. Gerontol. 40 , 939–947.)
I link yor question to my colleague Fabio Sgolastra who managed this issue in our group. Hope he can help.
To measure the HPGs, we dissected under a stereomicroscope, the honey bees’ heads and the hypopharyngeal glands were extracted in physiological Ringer solution. The glands were cleaned from muscles and aerial sacks, and afterwards coloured with Methylene blue dissolved in physiological solution. After this step, the HPGs were transferred to a glass slide with a concave basin filled with physiologic solution and covered with a cover slip (the presence of a concavity is necessary in order to avoid smashing up the glands with the cover slip). We measured the diameter of the acini under microscope (only the acini that were in horizontal position) and using a appropriate software (AxioVision 4.8).
Please find the following information: Honeybee heads were dissected in insect saline solution (Berger and Carmargo Abdalla 2005). Part of the HPGs were removed, placed in an electrophoresis stain (Coomassie brilliant blue dye R250) (Rhodes and Somerville, 2003) for 4 s and then on a microscopic slide (without a cover slip) to be photographed under a dissecting microscope. We used an image analysis system to capture and store the images, and later measure the diameters of the acini using Image Pro-Plus software. Acini diameter was measured twice and averaged.For more details see the publication of HATJINA et al. (2013). Sublethal doses of imidacloprid decreased size of hypopharyngeal glands and respiratory rhythm of honeybees in vivo. Apidologie, DOI 10.1007/s13592-013-0199-4
Good luck !
- Badges
- Science method
- Similar topics
- Biological Science
- Ecology