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2890Re: [beemonitoring] Identifying pollen collected from bees

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  • Peter Bernhardt
    Aug 21, 2013

      It's all relative.  I still think that acetolysis is overkill (and often counter-productive) if all you want to do is identify pollen carried by the animal visiting the flower vs. where the insect visited before it visited the host flower.  In my own case, I never count a pollen "species" taken from an insect as "present" unless I find a minimum of 25 identical grains or polyads (Linum, Acacia etc.) on the same slide.  It's a trick I developed in Australia.  I put non-flower visiting flies and beetles in the same killing jar as my acacia-visiting insects and counted the number of solitary grains the non-visitors carried due to contamination in the same jar.  The only "deal-breaker" should be the presence of a pollinarium (orchid or asclepioid) provided you can find viscidium or corpusculum still attached to the insect.   

      Of course, comparing pollen DNA extracted from the bee's crop seems a bit much but consider the future possibilities if the technology can be tweaked.  Eventually, it may tell you how many different trees (genotypes) of the same species were visited by the same bee before it was captured.  Based on another Australian project I noticed that some pollen-swallowing colletids invariably regurgitated their harvest if you killed them with fumes of ether or ethyl acetate.  Crop-derived grains are often a bit "chewed up" but are still identifiable following staining with Calberla's fluid.

      "What are you doing, Professor?"

      "I'm staining bee vomit."

      "Oh... that's nice, dear."  (It's always funnier with an Australian accent). 


      On Wed, Aug 21, 2013 at 2:39 PM, Jack Neff <jlnatctmi@...> wrote:

      The paper shows the method works but it seems like methodological overkill.  I would think one could distinguish silversword (Asteraceae) and pukiawe (Ericaceae) pollen by much simpler and cheaper methods than DNA analysis.  It is also not clear if this method gives quantitative results which is what one needs for diet analysis (not just presence or absence of various items).


      John L. Neff
      Central Texas Melittological Institute
      7307 Running Rope
      Austin,TX 78731 USA

      From: Colin Phifer <ccphifer@...>
      To: Bee United <beemonitoring@yahoogroups.com>
      Sent: Tuesday, August 20, 2013 7:57 PM
      Subject: Re: [beemonitoring] Identifying pollen collected from bees

      Another suggestion: you can now sequence the DNA for pollen ID. Here's an abstract from "Pollen foraging behaviour of solitary Hawaiian bees revealed through molecular pollen analysis" by Erin Wilson et al, published in Molecular Ecology vol 19 issue 21 pp  4428.

      "Obtaining quantitative information concerning pollinator behaviour has become a primary objective of pollination studies, but methodological limitations hinder progress towards this goal. Here, we use molecular genetic methods in an ecological context to demonstrate that endemic Hawaiian Hylaeus bees (Hymenoptera: Colletidae) selectively collect pollen from native plant species in Haleakala and Hawaii Volcanoes National Parks. We identified pollen DNA from the crops (internal storage organs) of 21 Hylaeusspecimens stored in ethanol for up to 3 years. Genetic analyses reveal high fidelity in pollen foraging despite the availability of pollen from multiple plant species present at each study site. At high elevations in Haleakala, pollen was available from more than 12 species of flowering plants, but Hawaiian silversword (Argyroxiphium sandwicense subsp. macrocephalum) comprised 86% of all pollen samples removed from bee crops. At lower elevations in both parks, we only detected pukiawe (Leptecophylla (Styphelia) tameiameiae) pollen inHylaeus crops despite the presence of other plant species in flower during our study. Furthermore, 100% of Hylaeus crops from which we successfully identified pollen contained native plant pollen. The molecular approaches developed in this study provide species-level information about floral visitation of Hawaiian Hylaeus that does not require specialized palynological expertise needed for high-throughput visual pollen identification. Building upon this approach, future studies can thus develop appropriate and customized criteria for assessing mixed pollen loads from a broader range of sources and from other global regions."

      I haven't tried this myself but it sounds very promising depending on your budget and timeline.

      • • • • • • • • • •
      Colin Phifer, PhD Student
      School of Forest Resources & Environmental Science
      Michigan Tech University
      1400 Townsend Drive
      Houghton, MI 49931-1295
      Phone: (808)-315-2830
      Email: ccphifer@...

      On Aug 20, 2013, at 8:02 PM, Linda Newstrom <newstroml@...> wrote:


      Hi Laura
      We struggled with not wanting to take the time and expense to do acetolysis to identify pollen for our project (www.treesforbeesnz.org ).
      We are investigating the nitrogen content of pollen from bee-collected pollen pellets and have found that to get accurate identifications
      we really had to do acetolysis and what a difference it makes. 
      But our project was focused on the pollen from pellets not a few grains on the honey bees.
      In any case, to identify unknown pollen a reference collection is best.  
      We are creating a reference collection based on both the acetolysed pollen and a slide made of unacetolysed pollen from fresh or frozen pollen stained in fuchsin jelly
      from the flowers and matching them to the acetolysed pollen from the bee pellets for confirmation of identity of the pollen.
      The unacetolysed counterparts from the flowers show us how far we can go with not acetolysing in terms of making accurate identifications
      Depends on the number of species and size of location you are dealing with.
      When it is impractical to do acetolysis we have a reference collection with both views.
      We solved the practicality problem by partnering up with a palynologist, Ian Raine,  at GNS Science, for identification of the pollen.
      The exchange of services and samples has been a win –win that serves both of our interests.
      See attached.
      Linda Newstrom-Lloyd
      From: beemonitoring@yahoogroups.com [mailto:beemonitoring@yahoogroups.com] On Behalf Of John Mola
      Sent: Tuesday, August 20, 2013 4:48 AM
      To: Doug Yanega
      Cc: Laura Russo; Bee United
      Subject: Re: [beemonitoring] Identifying pollen collected from bees
      I just wanted to back up a point Doug made: You only need a technique as detailed as the question you're asking. For me, I was going to avoid acetolysis but found it difficult to determine between Rubus and Malus pollens otherwise. If it had been simple to do that, or if I was asking a different question, it would have been fine to view the pollen untreated.

      Yes, acetolysis is rather time consuming and uses nasty chemicals, so if you can still make positive IDs without it, then go for it!
      On Mon, Aug 19, 2013 at 9:42 AM, Doug Yanega <dyanega@...> wrote:
      On 8/18/13 8:05 AM, Laura Russo wrote:
      Dear all,

      I am interested in identifying pollen samples collected from bees.  I have looked at some resources on the internet, but I thought I would probe this listserve to see if anyone has had experience with that process, and therefore recommendations for resources (i.e. books, websites, people, etc.).

      Any advice on the collection methodology would also be gratefully received.
      I'll offer my own experience in two techniques, one of which is an example of the lowest-tech option (which I imagine many here will scoff at), and you'll have to determine for yourself whether either is a viable approach in your case.

      When I was interested in this type of analysis, I took a course in palynology, and found the techniques - all of which centered around acetolysis - to be time-consuming, somewhat costly (I was doing research out-of-pocket), and poorly suited for very tiny quantities of pollen. Kearns & Inouye was not yet in print back in 1983, so I was flailing about a bit and had to devise my own approach.

      (1) Working with pinned museum specimens of bees, the number of pollen grains available can be very small, and the technique I used was to take an SEM stub with double-sided sticky tape and scrape pollen onto it directly. While these samples could not be examined under a light microscope, and while they didn't have the outer surfaces completely clean, they could still be viewed under the SEM just fine, if I could find someone who would let me have a few minutes of SEM time (which was infrequent, so I didn't get to make much use of this).

      (2) for wild-caught bees (whose behavior I was observing) I used regular scotch tape, rubbed the bee gently against a small piece of scotch tape (technically, "magic transparent" tape which can be written on) to produce a pollen smear, released the bee, and stuck the piece of tape to a microscope slide. I wrote the ID of the bee and the date and time on the piece of tape in pencil. The pollen was viewed by flipping the slide over. These samples were untreated, and that has drawbacks, but it took less than a minute to gather a sample, from catching the bee to having the sample mounted and labeled.

      In both cases, some reference images or samples are needed, using pollen grains that have been *similarly untreated* (no acetolysis). Perhaps things have changed since I did this work (back in the 1980's), but I found there to be very few publications with SEM photos of raw pollen grains, and there was (and probably still is) absolutely nothing published showing raw pollen under a light microscope. In the former case, I ended up with many unidentifiable pollen types, but for my purposes it was still enough to know whether a given bee was foraging on multiple pollen sources or not (i.e., I had a very specific question, and knowing the plant ID would've been great, but not essential for those samples). In the latter case, I systematically went around in the habitat in question and took samples of pollen directly from the anthers of all the flowering plants in bloom within about 100 meters of the nest site to create a reference collection. This actually proved to be very effective, allowing me to identify the source of over 80% of the collected samples with an extremely minimal amount of time, energy, or expense - OR disruption of the behaviors I was observing (most females would, after a short interval, return to their nest, deposit the pollen load from which I had removed a smear, and then resume foraging).

      I haven't stayed abreast of recent advances, so can't speak as to how comparable my solution is to the methods used by others. I will note that - out of curiosity - I just pulled out my slides and looked at them under a scope, and aside from strong fading of the color, they look pretty much the same as they did nearly 30 years ago when I collected them. Scotch tape is about as low-tech as one can get, and it looks pretty good as a research tool. ;-)


      Doug Yanega      Dept. of Entomology       Entomology Research Museum
      Univ. of California, Riverside, CA 92521-0314     skype: dyanega
      phone: (951) 827-4315 (disclaimer: opinions are mine, not UCR's)
        "There are some enterprises in which a careful disorderliness
              is the true method" - Herman Melville, Moby Dick, Chap. 82

      John Mola

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