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Identifying pollen collected from bees

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  • Laura Russo
    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
    Message 1 of 16 , Aug 18, 2013
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      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.

      Thank you very much for your time,
      Laura Russo

      --
      PhD Candidate
      Intercollege Graduate Degree Program in Ecology
      Biology Department
      Pennsylvania State University
      University Park, PA 16802

      office: 415 Mueller Lab
      phone: 814-865-7912
    • <barbara.abraham@...>
      Laura, We did it by collecting the pollen from blooming flowers and comparing it with the pollen on bees we collected on the flowers. The recipe for the gel
      Message 2 of 16 , Aug 19, 2013
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        Laura,

         

        We did it by collecting the pollen from blooming flowers and comparing it with the pollen on bees we collected on the  flowers. The recipe for the gel with stain is in Inouye's pollen methods book.

         

        Barb

         

        Barbara J Abraham, PhD

        Associate Professor

        SEEDS Chapter Advisor

        Department of Biological Sciences

        Hampton University

        Hampton, Va 23668

        barbara.abraham@...

        757-727-5283

         


        From: beemonitoring@yahoogroups.com [beemonitoring@yahoogroups.com] on behalf of Laura Russo [lar322@...]
        Sent: Sunday, August 18, 2013 11:05 AM
        To: Bee United
        Subject: [beemonitoring] Identifying pollen collected from bees

         

        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.

        Thank you very much for your time,
        Laura Russo

        --
        PhD Candidate
        Intercollege Graduate Degree Program in Ecology
        Biology Department
        Pennsylvania State University
        University Park, PA 16802

        office: 415 Mueller Lab
        phone: 814-865-7912

        The information contained in this message is intended only for the recipient, and may otherwise be privileged and confidential. If the reader of this message is not the intended recipient, or an employee or agent responsible for delivering this message to the intended recipient, please be aware that any dissemination or copying of this communication is strictly prohibited. If you have received this communication in error, please immediately notify us by replying to the message and deleting it from your computer. This footnote also confirms that this email has been scanned for all viruses by the Hampton University Center for Information Technology Enterprise Systems service.
      • John Mola
        Laura, What would be the goal of your sampling? Are you trying to determine proportions of the pollen they collected? Or just a list of the species regardless
        Message 3 of 16 , Aug 19, 2013
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          Laura,

          What would be the goal of your sampling? Are you trying to determine proportions of the pollen they collected? Or just a list of the species regardless of abundance?

          I ask because if you need proportions, then you need to mount slides in a way that allows you to count X amount of grains. If you just want to find out the different species you may have an "easier" time using SEM or some other powerful microscope technique.

          You may also find you need to acetolyze the pollen. Otherwise it can be very difficult to determine differences in ornamentation due to all the oils and whatnot clinging to the exine. Here's a basic protocol: http://www.dartmouth.edu/~emlab/manuals/sempreps/pollen.html    though you can email me personally for more detailed steps.

          Making a reference collection, as suggested above, is a definite step. Additionally, you have to be aware that each pollen "taxa" (since often you can only get to Genera) can look different depending on the plane it is oriented in. So you'll need to get good at determining the different appearances that the same species can have, recognizing that sometimes different orientations look like different species.

          It's tough to do, but can yield interesting results.

          Best of luck,

          John


          On Mon, Aug 19, 2013 at 6:48 AM, <barbara.abraham@...> wrote:
           

          Laura,

           

          We did it by collecting the pollen from blooming flowers and comparing it with the pollen on bees we collected on the  flowers. The recipe for the gel with stain is in Inouye's pollen methods book.

           

          Barb

           

          Barbara J Abraham, PhD

          Associate Professor

          SEEDS Chapter Advisor

          Department of Biological Sciences

          Hampton University

          Hampton, Va 23668

          barbara.abraham@...

          757-727-5283

           


          From: beemonitoring@yahoogroups.com [beemonitoring@yahoogroups.com] on behalf of Laura Russo [lar322@...]
          Sent: Sunday, August 18, 2013 11:05 AM
          To: Bee United
          Subject: [beemonitoring] Identifying pollen collected from bees

           

          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.

          Thank you very much for your time,
          Laura Russo

          --
          PhD Candidate
          Intercollege Graduate Degree Program in Ecology
          Biology Department
          Pennsylvania State University
          University Park, PA 16802

          office: 415 Mueller Lab
          phone: 814-865-7912

          The information contained in this message is intended only for the recipient, and may otherwise be privileged and confidential. If the reader of this message is not the intended recipient, or an employee or agent responsible for delivering this message to the intended recipient, please be aware that any dissemination or copying of this communication is strictly prohibited. If you have received this communication in error, please immediately notify us by replying to the message and deleting it from your computer. This footnote also confirms that this email has been scanned for all viruses by the Hampton University Center for Information Technology Enterprise Systems service.




          --
          John Mola
          JohnMMola@...




        • Jack Neff
          The basic method is as Barbara indicates, you make a reference collection of flowering plants in your area which you use to compare with pollen you find on
          Message 4 of 16 , Aug 19, 2013
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            The basic method is as Barbara indicates, you make a reference collection of flowering plants in your area which you use to compare with pollen you find on bees.  Unfortunately, species, generic and sometimes even family level ids of pollen are often quite diffcult unless you acetolylze your pollen, a rather nasty process which cleans up the grains the pollenkit so you can see the fine details of the exine.  SEM is now a very common way to go but again acetolysis is usually used to prepare the pollen.  Details and references are in "Practical Pollination Biology" by Dafni et al, 2005, Enviroquest Ltd and the older "Techniques for Pollination Biologists" by Kearns and Inouye, 1993 University of Colorado Press.

            best


            Jack
             
            John L. Neff
            Central Texas Melittological Institute
            7307 Running Rope
            Austin,TX 78731 USA
            512-345-7219


            From: "barbara.abraham@..." <barbara.abraham@...>
            To: lar322@...; beemonitoring@yahoogroups.com
            Sent: Monday, August 19, 2013 8:48 AM
            Subject: RE: [beemonitoring] Identifying pollen collected from bees

             
            Laura,
             
            We did it by collecting the pollen from blooming flowers and comparing it with the pollen on bees we collected on the  flowers. The recipe for the gel with stain is in Inouye's pollen methods book.
             
            Barb
             
            Barbara J Abraham, PhD
            Associate Professor
            SEEDS Chapter Advisor
            Department of Biological Sciences
            Hampton University
            Hampton, Va 23668
            757-727-5283
             

            From: beemonitoring@yahoogroups.com [beemonitoring@yahoogroups.com] on behalf of Laura Russo [lar322@...]
            Sent: Sunday, August 18, 2013 11:05 AM
            To: Bee United
            Subject: [beemonitoring] Identifying pollen collected from bees

             
            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.

            Thank you very much for your time,
            Laura Russo

            --
            PhD Candidate
            Intercollege Graduate Degree Program in Ecology
            Biology Department
            Pennsylvania State University
            University Park, PA 16802

            office: 415 Mueller Lab
            phone: 814-865-7912
            The information contained in this message is intended only for the recipient, and may otherwise be privileged and confidential. If the reader of this message is not the intended recipient, or an employee or agent responsible for delivering this message to the intended recipient, please be aware that any dissemination or copying of this communication is strictly prohibited. If you have received this communication in error, please immediately notify us by replying to the message and deleting it from your computer. This footnote also confirms that this email has been scanned for all viruses by the Hampton University Center for Information Technology Enterprise Systems service.


          • Doug Yanega
            ... 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
            Message 5 of 16 , Aug 19, 2013
            • 0 Attachment
              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. ;-)

              Peace,
              -- 
              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)
                           http://cache.ucr.edu/~heraty/yanega.html
                "There are some enterprises in which a careful disorderliness
                      is the true method" - Herman Melville, Moby Dick, Chap. 82
            • John Mola
              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
              Message 6 of 16 , Aug 19, 2013
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                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. ;-)

                Peace,
                -- 
                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)
                             http://cache.ucr.edu/~heraty/yanega.html
                  "There are some enterprises in which a careful disorderliness
                        is the true method" - Herman Melville, Moby Dick, Chap. 82




                --
                John Mola
                JohnMMola@...




              • Linda Newstrom
                Hi Laura We struggled with not wanting to take the time and expense to do acetolysis to identify pollen for our project
                Message 7 of 16 , Aug 20, 2013

                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.

                Thanks

                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. ;-)

                Peace,

                -- 
                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)
                             http://cache.ucr.edu/~heraty/yanega.html
                  "There are some enterprises in which a careful disorderliness
                        is the true method" - Herman Melville, Moby Dick, Chap. 82




                --

                John Mola
                JohnMMola@...






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              • Colin Phifer
                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
                Message 8 of 16 , Aug 20, 2013
                • 0 Attachment
                  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.

                  Thanks

                  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. ;-)

                  Peace,

                  -- 
                  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)
                               http://cache.ucr.edu/~heraty/yanega.html
                    "There are some enterprises in which a careful disorderliness
                          is the true method" - Herman Melville, Moby Dick, Chap. 82




                  --

                  John Mola
                  JohnMMola@...







                  Please consider the environment before printing this email
                  Warning: This electronic message together with any attachments is confidential. If you receive it in error: (i) you must not read, use, disclose, copy or retain it; (ii) please contact the sender immediately by reply email and then delete the emails.
                  The views expressed in this email may not be those of Landcare Research New Zealand Limited. http://www.landcareresearch.co.nz


                • Jack Neff
                  The paper shows the method works but it seems like methodological overkill.  I would think one could distinguish silversword (Asteraceae) and pukiawe
                  Message 9 of 16 , Aug 21, 2013
                  • 0 Attachment
                    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).

                    best

                    Jack
                     
                    John L. Neff
                    Central Texas Melittological Institute
                    7307 Running Rope
                    Austin,TX 78731 USA
                    512-345-7219


                    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.
                    Thanks
                    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. ;-)

                    Peace,

                    -- 
                    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)
                                 http://cache.ucr.edu/~heraty/yanega.html
                      "There are some enterprises in which a careful disorderliness
                            is the true method" - Herman Melville, Moby Dick, Chap. 82



                    --
                    John Mola
                    JohnMMola@...







                    Please consider the environment before printing this email
                    Warning: This electronic message together with any attachments is confidential. If you receive it in error: (i) you must not read, use, disclose, copy or retain it; (ii) please contact the sender immediately by reply email and then delete the emails.
                    The views expressed in this email may not be those of Landcare Research New Zealand Limited. http://www.landcareresearch.co.nz




                  • Peter Bernhardt
                    Jack: 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
                    Message 10 of 16 , Aug 21, 2013
                    • 0 Attachment
                      Jack:

                      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). 

                      Peter


                      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).

                      best

                      Jack
                       
                      John L. Neff
                      Central Texas Melittological Institute
                      7307 Running Rope
                      Austin,TX 78731 USA
                      512-345-7219


                      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.
                      Thanks
                      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. ;-)

                      Peace,

                      -- 
                      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)
                                   http://cache.ucr.edu/~heraty/yanega.html
                        "There are some enterprises in which a careful disorderliness
                              is the true method" - Herman Melville, Moby Dick, Chap. 82



                      --
                      John Mola
                      JohnMMola@...







                      Please consider the environment before printing this email
                      Warning: This electronic message together with any attachments is confidential. If you receive it in error: (i) you must not read, use, disclose, copy or retain it; (ii) please contact the sender immediately by reply email and then delete the emails.
                      The views expressed in this email may not be those of Landcare Research New Zealand Limited. http://www.landcareresearch.co.nz





                    • Karen W. Wright
                      I will be trying to identify pollen from scopae of Melissodes. I am applying for a grant to help pay for ITS sequencing for the pollen. With the DNA you can
                      Message 11 of 16 , Aug 21, 2013
                      • 0 Attachment
                        I will be trying to identify pollen from scopae of Melissodes. I am
                        applying for a grant to help pay for ITS sequencing for the pollen. With
                        the DNA you can measure genetic distance between host species which may be
                        a good index of polylecty. If I don't get the grant, it is back to slides
                        which is more time consuming, but cheaper, and less accurate (especially
                        within Asteraceae). My biggest disadvantage is that I don't have a local
                        library of ITS sequences since Melissodes' range is the New World. I will
                        have to depend on genbank for sequence identification. I think we are
                        getting to the point where there is enough available that this type of
                        barcoding is possible. Any thoughts on this would be appreciated. Thanks,
                        Karen


                        > Jack:
                        >
                        > 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).
                        >
                        > Peter
                        >
                        >
                        > 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).
                        >>
                        >> best
                        >>
                        >> Jack
                        >>
                        >> John L. Neff
                        >> Central Texas Melittological Institute
                        >> 7307 Running Rope
                        >> Austin,TX 78731 USA
                        >> 512-345-7219
                        >>
                        >> ------------------------------
                        >> *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.
                        >> Thanks
                        >> 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. ;-)
                        >>
                        >> Peace,
                        >>
                        >> --
                        >>
                        >> 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)
                        >>
                        >> http://cache.ucr.edu/~heraty/yanega.html
                        >>
                        >> "There are some enterprises in which a careful disorderliness
                        >>
                        >> is the true method" - Herman Melville, Moby Dick, Chap. 82
                        >>
                        >>
                        >>
                        >>
                        >> --
                        >> John Mola
                        >> JohnMMola@...
                        >>
                        >>
                        >>
                        >>
                        >>
                        >> ------------------------------
                        >>
                        >> Please consider the environment before printing this email
                        >> Warning: This electronic message together with any attachments is
                        >> confidential. If you receive it in error: (i) you must not read, use,
                        >> disclose, copy or retain it; (ii) please contact the sender immediately
                        >> by
                        >> reply email and then delete the emails.
                        >> The views expressed in this email may not be those of Landcare Research
                        >> New Zealand Limited. http://www.landcareresearch.co.nz
                        >>
                        >>
                        >>
                        >>
                        >>
                        >>
                        >
                      • T'ai Roulston
                        Karen: I m interested in knowing how you ll handle the quantitative question that Jack brought up. With Melissodes you should get a lot of unifloral pollen
                        Message 12 of 16 , Aug 21, 2013
                        • 0 Attachment
                          Karen:

                          I'm interested in knowing how you'll handle the quantitative question that Jack brought up. With Melissodes you should get a lot of unifloral pollen loads but are you going to examine pollen loads with a microscope to know the relative amounts collected when you hit mixed loads? Or are there good molecular protocols to handle this now?

                          If you are still interested in Melissodes specimens, I could provide a few eastern ones.

                          T'ai
                          On Aug 21, 2013, at 5:46 PM, Karen W. Wright wrote:

                           

                          I will be trying to identify pollen from scopae of Melissodes. I am
                          applying for a grant to help pay for ITS sequencing for the pollen. With
                          the DNA you can measure genetic distance between host species which may be
                          a good index of polylecty. If I don't get the grant, it is back to slides
                          which is more time consuming, but cheaper, and less accurate (especially
                          within Asteraceae). My biggest disadvantage is that I don't have a local
                          library of ITS sequences since Melissodes' range is the New World. I will
                          have to depend on genbank for sequence identification. I think we are
                          getting to the point where there is enough available that this type of
                          barcoding is possible. Any thoughts on this would be appreciated. Thanks,
                          Karen

                          > Jack:
                          >
                          > 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).
                          >
                          > Peter
                          >
                          >
                          > 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).
                          >>
                          >> best
                          >>
                          >> Jack
                          >>
                          >> John L. Neff
                          >> Central Texas Melittological Institute
                          >> 7307 Running Rope
                          >> Austin,TX 78731 USA
                          >> 512-345-7219
                          >>
                          >> ------------------------------
                          >> *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.
                          >> Thanks
                          >> 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. ;-)
                          >>
                          >> Peace,
                          >>
                          >> --
                          >>
                          >> 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)
                          >>
                          >> http://cache.ucr.edu/~heraty/yanega.html
                          >>
                          >> "There are some enterprises in which a careful disorderliness
                          >>
                          >> is the true method" - Herman Melville, Moby Dick, Chap. 82
                          >>
                          >>
                          >>
                          >>
                          >> --
                          >> John Mola
                          >> JohnMMola@...
                          >>
                          >>
                          >>
                          >>
                          >>
                          >> ------------------------------
                          >>
                          >> Please consider the environment before printing this email
                          >> Warning: This electronic message together with any attachments is
                          >> confidential. If you receive it in error: (i) you must not read, use,
                          >> disclose, copy or retain it; (ii) please contact the sender immediately
                          >> by
                          >> reply email and then delete the emails.
                          >> The views expressed in this email may not be those of Landcare Research
                          >> New Zealand Limited. http://www.landcareresearch.co.nz
                          >>
                          >>
                          >>
                          >>
                          >>
                          >>
                          >


                          T'ai Roulston
                          Curator, State Arboretum of Virginia
                          Research Assoc. Prof., Dept of Envi. Sci.
                          University of Virginia



                        • Brosi, Berry J
                          Hi all, The Wilson et al. paper does not give a protocol for sequencing mixed pollen loads from bees. It can only return one sequence. Thus, it will give you
                          Message 13 of 16 , Aug 22, 2013
                          • 0 Attachment

                            Hi all,

                            The Wilson et al. paper does not give a protocol for sequencing mixed pollen loads from bees.  It can only return one sequence.  Thus, it will give you a (single) sequence if there is a single pollen type in a pollen load, or a single sequence with some "indeterminate" base pair reads if there is a mixed pollen load with one species really dominating, or will return nothing if you have a very diverse pollen load (or if there is just a sequencing error!).  Such an analysis can work passably (marginally?) in a plant-species-poor area, especially when working with pollinators that are likely to visit a limited subset of plants, e.g. the Hawaiian Hylaeus that Wilson et al. studied.  But it is unlikely to work well in most other systems, e.g. which will have greater plant species richness and pollinators that are likely to visit multiple plant species.

                            In addition, information on pollen quantities in a pollen load *cannot* be determined with molecular analyses currently.  DNA barcoding methods use sequences on organellar genomes (chloroplast, ribosome, etc.) because they are less subject to recombination than nuclear markers.  Copy number of organellar genomes is known to vary considerably—I don't know the specifics but I believe we are talking orders of magnitude—in pollen of different plant taxa, so even if one were to use quantitative PCR, etc., having greater relative quantities of PCR product of pollen from plant species A versus pollen of plant species B would not even necessarily tell you that species A has more pollen grains than B in your sample.

                            Finally, unfortunately for most areas, there is very limited coverage of DNA barcoding markers for plants on GenBank, with the exception of a few areas that have been intensively studied for exactly that purpose.

                            cheers,
                            Berry

                            -----------------------------------------------------------
                            Berry J. Brosi, Ph.D.
                            Assistant Professor
                            Department of Environmental Studies
                            Emory University
                            http://www.envs.emory.edu/faculty/brosi/brosilab/home.html


                            On Aug 21, 2013, at 7:28 PM, T'ai Roulston <thr8z@...>
                             wrote:

                             

                            Karen:


                            I'm interested in knowing how you'll handle the quantitative question that Jack brought up. With Melissodes you should get a lot of unifloral pollen loads but are you going to examine pollen loads with a microscope to know the relative amounts collected when you hit mixed loads? Or are there good molecular protocols to handle this now?

                            If you are still interested in Melissodes specimens, I could provide a few eastern ones.

                            T'ai
                            On Aug 21, 2013, at 5:46 PM, Karen W. Wright wrote:

                             

                            I will be trying to identify pollen from scopae of Melissodes. I am
                            applying for a grant to help pay for ITS sequencing for the pollen. With
                            the DNA you can measure genetic distance between host species which may be
                            a good index of polylecty. If I don't get the grant, it is back to slides
                            which is more time consuming, but cheaper, and less accurate (especially
                            within Asteraceae). My biggest disadvantage is that I don't have a local
                            library of ITS sequences since Melissodes' range is the New World. I will
                            have to depend on genbank for sequence identification. I think we are
                            getting to the point where there is enough available that this type of
                            barcoding is possible. Any thoughts on this would be appreciated. Thanks,
                            Karen

                            > Jack:
                            >
                            > 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).
                            >
                            > Peter
                            >
                            >
                            > 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).
                            >>
                            >> best
                            >>
                            >> Jack
                            >>
                            >> John L. Neff
                            >> Central Texas Melittological Institute
                            >> 7307 Running Rope
                            >> Austin,TX 78731 USA
                            >> 512-345-7219
                            >>
                            >> ------------------------------
                            >> *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.
                            >> Thanks
                            >> 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. ;-)
                            >>
                            >> Peace,
                            >>
                            >> --
                            >>
                            >> 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)
                            >>
                            >> http://cache.ucr.edu/~heraty/yanega.html
                            >>
                            >> "There are some enterprises in which a careful disorderliness
                            >>
                            >> is the true method" - Herman Melville, Moby Dick, Chap. 82
                            >>
                            >>
                            >>
                            >>
                            >> --
                            >> John Mola
                            >> JohnMMola@...
                            >>
                            >>
                            >>
                            >>
                            >>
                            >> ------------------------------
                            >>
                            >> Please consider the environment before printing this email
                            >> Warning: This electronic message together with any attachments is
                            >> confidential. If you receive it in error: (i) you must not read, use,
                            >> disclose, copy or retain it; (ii) please contact the sender immediately
                            >> by
                            >> reply email and then delete the emails.
                            >> The views expressed in this email may not be those of Landcare Research
                            >> New Zealand Limited. http://www.landcareresearch.co.nz
                            >>
                            >>
                            >>
                            >>
                            >>
                            >>
                            >


                            T'ai Roulston
                            Curator, State Arboretum of Virginia
                            Research Assoc. Prof., Dept of Envi. Sci.
                            University of Virginia








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                          • Peter Bernhardt
                            Dear Berry: Hmmmm. I ll bet it would work very nicely for pollinia carried by bees, though. Each pollinium consists exclusively of pollen from one flower.
                            Message 14 of 16 , Aug 23, 2013
                            • 0 Attachment
                              Dear Berry:

                              Hmmmm.  I'll bet it would work very nicely for pollinia carried by bees, though.  Each pollinium consists exclusively of pollen from one flower.  Some orchid pollinaria of the Neotropics can be identified to genus on the basis of morphology and where it is deposited on the bee's body.  The analyst knows he/she is removing pollen of Stanhopea but may not know which Stanhopea spp.  You technique will answer that question.

                              Likewise, this might also be applied to milkweeds (Asclepias) and their allies.  They also produce pollinaria and, based many past studies, a bee may visit more than one Asclepias spp. in bloom during the same foraging bout.  Pollinaria of milkweeds are hard to identify once they leave the flower, and different Asclepias app. deposit the pollinaria on the same bee legs, so your analysis would make it possible to find out whether the six pollinaria on the same bee belonged to the same species.  Of course, it would be best to test each pollinarium one at a time...right?

                              Peter

                              Peter  


                              On Thu, Aug 22, 2013 at 2:05 PM, Brosi, Berry J <bbrosi@...> wrote:
                               


                              Hi all,

                              The Wilson et al. paper does not give a protocol for sequencing mixed pollen loads from bees.  It can only return one sequence.  Thus, it will give you a (single) sequence if there is a single pollen type in a pollen load, or a single sequence with some "indeterminate" base pair reads if there is a mixed pollen load with one species really dominating, or will return nothing if you have a very diverse pollen load (or if there is just a sequencing error!).  Such an analysis can work passably (marginally?) in a plant-species-poor area, especially when working with pollinators that are likely to visit a limited subset of plants, e.g. the Hawaiian Hylaeus that Wilson et al. studied.  But it is unlikely to work well in most other systems, e.g. which will have greater plant species richness and pollinators that are likely to visit multiple plant species.

                              In addition, information on pollen quantities in a pollen load *cannot* be determined with molecular analyses currently.  DNA barcoding methods use sequences on organellar genomes (chloroplast, ribosome, etc.) because they are less subject to recombination than nuclear markers.  Copy number of organellar genomes is known to vary considerably—I don't know the specifics but I believe we are talking orders of magnitude—in pollen of different plant taxa, so even if one were to use quantitative PCR, etc., having greater relative quantities of PCR product of pollen from plant species A versus pollen of plant species B would not even necessarily tell you that species A has more pollen grains than B in your sample.

                              Finally, unfortunately for most areas, there is very limited coverage of DNA barcoding markers for plants on GenBank, with the exception of a few areas that have been intensively studied for exactly that purpose.

                              cheers,
                              Berry

                              -----------------------------------------------------------
                              Berry J. Brosi, Ph.D.
                              Assistant Professor
                              Department of Environmental Studies
                              Emory University
                              http://www.envs.emory.edu/faculty/brosi/brosilab/home.html


                              On Aug 21, 2013, at 7:28 PM, T'ai Roulston <thr8z@...>
                               wrote:

                               

                              Karen:


                              I'm interested in knowing how you'll handle the quantitative question that Jack brought up. With Melissodes you should get a lot of unifloral pollen loads but are you going to examine pollen loads with a microscope to know the relative amounts collected when you hit mixed loads? Or are there good molecular protocols to handle this now?

                              If you are still interested in Melissodes specimens, I could provide a few eastern ones.

                              T'ai
                              On Aug 21, 2013, at 5:46 PM, Karen W. Wright wrote:

                               

                              I will be trying to identify pollen from scopae of Melissodes. I am
                              applying for a grant to help pay for ITS sequencing for the pollen. With
                              the DNA you can measure genetic distance between host species which may be
                              a good index of polylecty. If I don't get the grant, it is back to slides
                              which is more time consuming, but cheaper, and less accurate (especially
                              within Asteraceae). My biggest disadvantage is that I don't have a local
                              library of ITS sequences since Melissodes' range is the New World. I will
                              have to depend on genbank for sequence identification. I think we are
                              getting to the point where there is enough available that this type of
                              barcoding is possible. Any thoughts on this would be appreciated. Thanks,
                              Karen

                              > Jack:
                              >
                              > 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).
                              >
                              > Peter
                              >
                              >
                              > 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).
                              >>
                              >> best
                              >>
                              >> Jack
                              >>
                              >> John L. Neff
                              >> Central Texas Melittological Institute
                              >> 7307 Running Rope
                              >> Austin,TX 78731 USA
                              >> 512-345-7219
                              >>
                              >> ------------------------------
                              >> *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.
                              >> Thanks
                              >> 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. ;-)
                              >>
                              >> Peace,
                              >>
                              >> --
                              >>
                              >> 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)
                              >>
                              >> http://cache.ucr.edu/~heraty/yanega.html
                              >>
                              >> "There are some enterprises in which a careful disorderliness
                              >>
                              >> is the true method" - Herman Melville, Moby Dick, Chap. 82
                              >>
                              >>
                              >>
                              >>
                              >> --
                              >> John Mola
                              >> JohnMMola@...
                              >>
                              >>
                              >>
                              >>
                              >>
                              >> ------------------------------
                              >>
                              >> Please consider the environment before printing this email
                              >> Warning: This electronic message together with any attachments is
                              >> confidential. If you receive it in error: (i) you must not read, use,
                              >> disclose, copy or retain it; (ii) please contact the sender immediately
                              >> by
                              >> reply email and then delete the emails.
                              >> The views expressed in this email may not be those of Landcare Research
                              >> New Zealand Limited. http://www.landcareresearch.co.nz
                              >>
                              >>
                              >>
                              >>
                              >>
                              >>
                              >


                              T'ai Roulston
                              Curator, State Arboretum of Virginia
                              Research Assoc. Prof., Dept of Envi. Sci.
                              University of Virginia








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                            • Berry Brosi
                              I agree, it should work quite well for pollinia, and yes, if following the Wilson et al. protocol each pollinium should be analyzed separately. The issue that
                              Message 15 of 16 , Aug 23, 2013
                              • 0 Attachment

                                I agree, it should work quite well for pollinia, and yes, if following the Wilson et al. protocol each pollinium should be analyzed separately.  The issue that we don't have good coverage of plants in terms of representation in GenBank remains, and is likely particularly acute for tropical orchids.

                                Cheers,
                                Berry


                                -----------------------------------------------------------
                                Berry J. Brosi, Ph.D.
                                Assistant Professor
                                Department of Environmental Studies
                                Emory University


                                On Aug 23, 2013, at 10:39 AM, Peter Bernhardt <bernhap2@...> wrote:

                                Dear Berry:

                                Hmmmm.  I'll bet it would work very nicely for pollinia carried by bees, though.  Each pollinium consists exclusively of pollen from one flower.  Some orchid pollinaria of the Neotropics can be identified to genus on the basis of morphology and where it is deposited on the bee's body.  The analyst knows he/she is removing pollen of Stanhopea but may not know which Stanhopea spp.  You technique will answer that question.

                                Likewise, this might also be applied to milkweeds (Asclepias) and their allies.  They also produce pollinaria and, based many past studies, a bee may visit more than one Asclepias spp. in bloom during the same foraging bout.  Pollinaria of milkweeds are hard to identify once they leave the flower, and different Asclepias app. deposit the pollinaria on the same bee legs, so your analysis would make it possible to find out whether the six pollinaria on the same bee belonged to the same species.  Of course, it would be best to test each pollinarium one at a time...right?

                                Peter

                                Peter  


                                On Thu, Aug 22, 2013 at 2:05 PM, Brosi, Berry J <bbrosi@...> wrote:
                                 


                                Hi all,

                                The Wilson et al. paper does not give a protocol for sequencing mixed pollen loads from bees.  It can only return one sequence.  Thus, it will give you a (single) sequence if there is a single pollen type in a pollen load, or a single sequence with some "indeterminate" base pair reads if there is a mixed pollen load with one species really dominating, or will return nothing if you have a very diverse pollen load (or if there is just a sequencing error!).  Such an analysis can work passably (marginally?) in a plant-species-poor area, especially when working with pollinators that are likely to visit a limited subset of plants, e.g. the Hawaiian Hylaeus that Wilson et al. studied.  But it is unlikely to work well in most other systems, e.g. which will have greater plant species richness and pollinators that are likely to visit multiple plant species.

                                In addition, information on pollen quantities in a pollen load *cannot* be determined with molecular analyses currently.  DNA barcoding methods use sequences on organellar genomes (chloroplast, ribosome, etc.) because they are less subject to recombination than nuclear markers.  Copy number of organellar genomes is known to vary considerably—I don't know the specifics but I believe we are talking orders of magnitude—in pollen of different plant taxa, so even if one were to use quantitative PCR, etc., having greater relative quantities of PCR product of pollen from plant species A versus pollen of plant species B would not even necessarily tell you that species A has more pollen grains than B in your sample.

                                Finally, unfortunately for most areas, there is very limited coverage of DNA barcoding markers for plants on GenBank, with the exception of a few areas that have been intensively studied for exactly that purpose.

                                cheers,
                                Berry

                                -----------------------------------------------------------
                                Berry J. Brosi, Ph.D.
                                Assistant Professor
                                Department of Environmental Studies
                                Emory University
                                http://www.envs.emory.edu/faculty/brosi/brosilab/home.html


                                On Aug 21, 2013, at 7:28 PM, T'ai Roulston <thr8z@...>
                                 wrote:

                                 

                                Karen:


                                I'm interested in knowing how you'll handle the quantitative question that Jack brought up. With Melissodes you should get a lot of unifloral pollen loads but are you going to examine pollen loads with a microscope to know the relative amounts collected when you hit mixed loads? Or are there good molecular protocols to handle this now?

                                If you are still interested in Melissodes specimens, I could provide a few eastern ones.

                                T'ai
                                On Aug 21, 2013, at 5:46 PM, Karen W. Wright wrote:

                                 

                                I will be trying to identify pollen from scopae of Melissodes. I am
                                applying for a grant to help pay for ITS sequencing for the pollen. With
                                the DNA you can measure genetic distance between host species which may be
                                a good index of polylecty. If I don't get the grant, it is back to slides
                                which is more time consuming, but cheaper, and less accurate (especially
                                within Asteraceae). My biggest disadvantage is that I don't have a local
                                library of ITS sequences since Melissodes' range is the New World. I will
                                have to depend on genbank for sequence identification. I think we are
                                getting to the point where there is enough available that this type of
                                barcoding is possible. Any thoughts on this would be appreciated. Thanks,
                                Karen

                                > Jack:
                                >
                                > 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).
                                >
                                > Peter
                                >
                                >
                                > 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).
                                >>
                                >> best
                                >>
                                >> Jack
                                >>
                                >> John L. Neff
                                >> Central Texas Melittological Institute
                                >> 7307 Running Rope
                                >> Austin,TX 78731 USA
                                >> 512-345-7219
                                >>
                                >> ------------------------------
                                >> *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.
                                >> Thanks
                                >> 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. ;-)
                                >>
                                >> Peace,
                                >>
                                >> --
                                >>
                                >> 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)
                                >>
                                >> http://cache.ucr.edu/~heraty/yanega.html
                                >>
                                >> "There are some enterprises in which a careful disorderliness
                                >>
                                >> is the true method" - Herman Melville, Moby Dick, Chap. 82
                                >>
                                >>
                                >>
                                >>
                                >> --
                                >> John Mola
                                >> JohnMMola@...
                                >>
                                >>
                                >>
                                >>
                                >>
                                >> ------------------------------
                                >>
                                >> Please consider the environment before printing this email
                                >> Warning: This electronic message together with any attachments is
                                >> confidential. If you receive it in error: (i) you must not read, use,
                                >> disclose, copy or retain it; (ii) please contact the sender immediately
                                >> by
                                >> reply email and then delete the emails.
                                >> The views expressed in this email may not be those of Landcare Research
                                >> New Zealand Limited. http://www.landcareresearch.co.nz
                                >>
                                >>
                                >>
                                >>
                                >>
                                >>
                                >


                                T'ai Roulston
                                Curator, State Arboretum of Virginia
                                Research Assoc. Prof., Dept of Envi. Sci.
                                University of Virginia








                                This e-mail message (including any attachments) is for the sole use of
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                              • Elliott
                                Hello all, Very interesting thread! I love it! Has anyone used Rex Sawyer s Pollen Identification for Beekeepers ? I have ordered it and and I am not sure how
                                Message 16 of 16 , Aug 23, 2013
                                • 0 Attachment
                                  Hello all,
                                  Very interesting thread! I love it!
                                  Has anyone used Rex Sawyer's "Pollen Identification for Beekeepers"? I have ordered it and and I am not sure how practical or applicable it is. Very nice piece of work though, but I am still floundering and tiring to identify what I am looking at that I have collected.

                                  Also, has anyone looked at this online book: An introduction to Pollen Analysis? Here is it's link: http://archive.org/details/introductiontopo029798mbp

                                  Cheers,
                                  EF
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