We thank Eli Jensen, Stefano Padilla, and Kasey Yturralde for assistance, and Karen London and anonymous reviewers of the manuscript. We thank Jake Baker and Karla Torres for video footage. Funding was provided to R.W.H by the NAU School of Forestry and NAU Technology and Research Initiative Fund (TRIF).

1. Phloem Selection and Removal from Tree
<ol>
	<li>Select a tree with particular characteristics. Collect phloem from pines (i.e., trees in the genus Pinus) as they have a distinctive phloem layer that is several millimeters thick.18,27 Once a tree is located that has few lower branches, inspect it for any other defects such as insect attacks and/or pathogens. Alternatively, use phloem from other conifer trees such as spruce trees in phloem sandwiches.9 Other trees species may be suitable for phloem removal such as hardwoods. 
	NOTE: Coniferous trees with large crowns typically have the thickest phloem tissue. To maximize the amount of phloem, it is best to cut trees that have few defects and branches on the trunk (bole). Pine species that are self-pruning work best. Phloem is typically thickest during the growing season and is thicker higher up the trunk than near the ground. Phloem is sometimes difficult to remove from trees during the fall or winter seasons.</li>
	<li>Cut a tree down or use recently cut logs to obtain phloem tissue. Choose a falling direction that will minimize damage to the tree as well as nearby trees. Cut branches off the trunk to allow easier access to bark materials during phloem removal.</li>
	<li>Once a tree is cut (felled; dropped on the ground) begin scraping the bark off the trunk with a sharp draw blade (Fig. 1A). Scrape an area of the bark off until the phloem layer is reached. 
	NOTE: The phloem layer is typically lighter in color (e.g., cream color) and moist, while the outer bark is drier and darker in color (Fig. 1B). Take special care not to scrape the phloem. The area of bark scraped depends upon the size of the phloem piece(s) required.</li>
	<li>After the bark is removed, cut an outline of the phloem piece with a sharp knife. Make sure the knife cuts all the way to the xylem (Fig. 2A).</li>
	<li>To remove the phloem, start at a corner of the phloem piece using fingers to carefully peel back the phloem. Use a knife to assist in scraping the phloem off the xylem. Continue pulling the phloem off until the entire piece is removed. 
	NOTE: Phloem typically peels off of the tree easiest in the summer. For phloem that is exceedingly difficult to remove, a spatula-shaped tool can aid in the effort.</li>
	<li>Place the phloem piece immediately into a sterile bag. For best results, vacuum seal the bag (Fig. 2B) or, if using ziploc bags, remove all air from the bag. This increases the longevity of the phloem. Optionally, place multiple pieces of phloem in one bag. Store phloem in airtight bags slightly above freezing (between 1 and 10C) to retain its freshness.</li>
</ol>
2. Creating the Phloem Sandwich
<ol>
	<li>Cut two equal pieces of clear acrylic, polycarbonate, or a similar hard, clear material (e.g., glass) slightly larger than the piece of phloem (Fig. 2C). Round the edges of the acrylic to prevent the corners from tearing the Parafilm seal. This protocol uses a 1/8 in. thick clear acrylic. 
	NOTE: The size of the cut pieces depends on the needs of the subject organism and the length of the study. For instance, a pair of bark beetles utilizes 4 dm2 of phloem over a month period, but will only need 1 dm2 if the study takes place within several days.</li>
	<li>Drill a hole into one of the acrylic pieces to allow entry of study organism(s). The size and number of holes depends on the objectives (Fig. 2D).</li>
	<li>Prior to placing the phloem between the acrylic pieces, sterilize the acrylic surface (with &#62; 70% ethanol) or, if using new acrylic, remove the protective film.</li>
	<li>Place the fresh piece of phloem between the sterile acrylic pieces. Orient the acrylic piece with the hole(s) on the inner or outer side of the phloem, whichever is needed. Typically, face the outer side of the phloem (side that had bark on it) toward the entry holes.</li>
</ol>
3. Sealing the Phloem Sandwich
<ol>
	<li>To create a temporary seal around the phloem sandwich, use 2 in. wide Parafilm strips pulled around the edges of the phloem sandwich (Fig. 2C). Alternately, use a polyvinylidene chloride wrap to seal the edges.6 Next, place a clamp on each side of the sandwich to squeeze the acrylic to the phloem. Make sure the entire surface is clamped down to prevent air space between the acrylic and phloem (Fig 2C). If not clamped properly, specimens may move in between the acrylic and phloem.</li>
	<li>To create a semi-permanent seal add a non-gluing epoxy or petroleum jelly around the phloem. Make sure that the material completely surrounds the phloem. Next, use clamps or screws with bolts (may need to drill holes ahead of time) to hold the acrylic tight to the phloem. The sandwiches only remain viable for up to one or two months.</li>
	<li>Depending on the oxygen needs of the study organisms, add an air filter on one or more sides of the sandwich. This will allow air to enter into the phloem sandwich but limit water loss from the phloem. We use simple charcoal filters that also reduce the risk of fungal and bacterial contamination.</li>
	<li>For study specimens that require entry and exit at will, replace one of the acrylic pieces with wood or similar material that an insect can bore into. This is particularly important for the observation of wood borers, because after their larval state is completed in the phloem layer, they then bore into the xylem.</li>
	<li>To prevent test subjects from exiting the entry holes, place small petri dishes (or other objects, tape) over the holes, blocking escape. Since organisms that reside in these spaces are accustom to low light levels, it may be necessary to place the sandwiches in a dark room or box, or place opaque material on top to block the light.</li>
</ol>
4. Observing Organisms in the Phloem Sandwich
<ol>
	<li>Introduce study specimens into entry hole of the phloem sandwich (Fig. 2D). Observe specimens using a dissecting microscope under a red light or white light set at a low setting (Fig. 2E).</li>
	<li>To record the activities or growth of specimens within the phloem sandwich attach a camera or video camera to the microscope. Attach special video cameras to the microscope eyepiece (Fig. 2E &#38; F). For very small organisms such as mites, nematodes, and pseudoscorpions, use a high definition video camera attached to the microscope.</li>
	<li>To record sounds, insert microphones into the entry hole of the phloem sandwich or through the side of the phloem sandwich. Because the entry hole is small, use a small microphone such as an electret condenser microphone.</li>
	<li>To record from the side or surface of the phloem sandwich use a piezo element (Fig. 3B). To playback sound, use the same piezo element or attach a tactile transducer (excitor) to the surface of the top or bottom of the acrylic.</li>
</ol>

<ol>
	<li>Aukema, B. H., Raffa, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+adult+and+larval+Platysoma+cylindrical.+(Coleoptera:+Histeridae)+and+larval+Medetera+bistriata.+(Diptera:+Dolichopodidae)+during+subcortical+predation+of+Ips+pini+(Coleoptera:+Scolytidae).">Behavior of adult and larval Platysoma cylindrical. (Coleoptera: Histeridae) and larval Medetera bistriata. (Diptera: Dolichopodidae) during subcortical predation of Ips pini (Coleoptera: Scolytidae).</a> J Insect Behav. 17, 115-128 (2004).</li><li>Bedard, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+number+of+larval+instars+and+the+appropriate+length+of+the+larval+stadia+of+Dendroctonus+pseudotsugae.+Hopk.,+with+a+method+for+their+determination+in+relation+to+other+bark+beetle.">The number of larval instars and the appropriate length of the larval stadia of Dendroctonus pseudotsugae. Hopk., with a method for their determination in relation to other bark beetle.</a> J Econ Entomol. 26, 128-134 (1933).</li><li>Bridges, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen-fixing+bacteria+associated+with+bark+beetles.">Nitrogen-fixing bacteria associated with bark beetles.</a> Microb Ecol. 7, 131-137 (1981).</li><li>Cardoza, Y. J., Klepzig, K. D., Raffa, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+in+oral+secretions+of+an+endophytic+insect+inhibit+antagonistic+fungi.">Bacteria in oral secretions of an endophytic insect inhibit antagonistic fungi.</a> Ecol Entomol. 31, 636-635 (2006).</li><li>Cardoza, Y. J., Moser, J. C., Klepzig, K. D., Raffa, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipartite+symbioses+among+fungi,+mites,+nematodes,+and+the+spruce+beetle,+Dendroctonus+rufipennis.">Multipartite symbioses among fungi, mites, nematodes, and the spruce beetle, Dendroctonus rufipennis.</a> Environ Entomol. 37, 956-963 (2008).</li><li>Chen, H. -. F., Salcedo, C., Sun, J. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+mate+choice+by+chemical+cues+leads+to+higher+reproductive+success+in+a+bark+beetle.">Male mate choice by chemical cues leads to higher reproductive success in a bark beetle.</a> Animal Behavior. 83, 421-427 (2012).</li><li>Dodds, K. J., Graber, C., Stephen, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facultative+intra+guild+predation+by+larval+Cerambycidae+(Coleoptera)+on+bark+beetle+larvae+(Coleoptera:+Scolytidae).">Facultative intra guild predation by larval Cerambycidae (Coleoptera) on bark beetle larvae (Coleoptera: Scolytidae).</a> Environmental Entomology. 30, 17-22 (2001).</li><li>Franklin, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+studying+the+insect+parasites+of+Dendroctonus+frontalis.+and+other+bark+beetles+(Coleoptera:+Scolytidae).">A technique for studying the insect parasites of Dendroctonus frontalis. and other bark beetles (Coleoptera: Scolytidae).</a> Journal of Georgia Entomology Society. 2, 43-44 (1967).</li><li>Gries, G., Pierce, H. D., Lindgren, B. S., Borden, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+techniques+for+capturing+and+analyzing+semiochemicals+for+Scolytid+beetles+(Coleoptera:+Scolytidae).">New techniques for capturing and analyzing semiochemicals for Scolytid beetles (Coleoptera: Scolytidae).</a> J Econ Entomol. 81, 1715-1720 (1988).</li><li>Hofstetter, R. W., Moser, J. C., McGuire, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+of+the+mite+Schizosthetus+lyriformis.+(Acari:+Parasitidae)+preying+on+bark+beetle+eggs+and+larvae.">Observations of the mite Schizosthetus lyriformis. (Acari: Parasitidae) preying on bark beetle eggs and larvae.</a> Entomology. News. 120, 397-400 (2009).</li><li>Hofstetter, R. W., Klepzig, K. D., Coulson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11:+Mutualists+and+Phoronts+of+the+Southern+Pine+Beetle.">Chapter 11: Mutualists and Phoronts of the Southern Pine Beetle.</a> United States Dept. of Agriculture Forest Service, Southern Research Station General Technical Report SRS-140. , 161-181 (2011).</li><li>Hofstetter, R. W., Dunn, D. D., McGuire, R., Potter, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+acoustic+technology+to+reduce+bark+beetle+reproduction).">Using acoustic technology to reduce bark beetle reproduction).</a> Pest Manag Sci. 70, 24-27 (2014).</li><li>Hopping, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+rearing+Ips.">Techniques for rearing Ips.</a> De Geer (Coleoptera: Scolytidae). Can Entomol. 93, 1050-1063 (1961).</li><li>Hougardy, E., Gregoire, J. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleptoparasitism+increases+the+host+finding+ability+of+a+polyphagous+parasitoid+species,+Rhopalicus+tutela+(Hymenoptera:+Pteromalidae).">Cleptoparasitism increases the host finding ability of a polyphagous parasitoid species, Rhopalicus tutela (Hymenoptera: Pteromalidae).</a> Behav Ecol Sociobiol. 55, 184-189 (2003).</li><li>Kaston, B. J., Riggs, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+larvae+of+the+native+elm+bark+beetle.">Studies on the larvae of the native elm bark beetle.</a> J Econ Entomol. 30, 98-108 (1937).</li><li>Kinn, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+of+Dendrolaelaps+neodisetus.+(Mesostigmata:+Digamasellidae),+a+nematophagous+mite+associated+with+pine+bark+beetles+(Coleoptera:+Scolytidae).">Life cycle of Dendrolaelaps neodisetus. (Mesostigmata: Digamasellidae), a nematophagous mite associated with pine bark beetles (Coleoptera: Scolytidae).</a> Environ Entomol. 13, 1141-1144 (1984).</li><li>Kinn, D. N., Miller, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phloem+sandwich+unit+for+observing+bark+beetles,+associated+predators,+and+parasites.">A phloem sandwich unit for observing bark beetles, associated predators, and parasites.</a> USDA FS Res. Notes SO-269. , 3 (1981).</li><li>Lieutier, F., Day, K. R., Battisti, A., Gregoire, J. -. C., Evans, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bark and wood boring insects in living trees in Europe, a synthesis. , 569 (2004).</li><li>Massey, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+nematode+parasites+and+associates+on+bark+beetles+in+the+United+States.">The influence of nematode parasites and associates on bark beetles in the United States.</a> Bulletin of the Entomology Society of America. 12, 384-386 (1966).</li><li>Moser, J. C., Roton, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mites+associated+with+southern+pine+bark+beetles+in.">Mites associated with southern pine bark beetles in.</a> Allen Parish, Louisiana. Can Entomol. 103, 1775-1798 (1971).</li><li>Mills, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+enemies+of+scolytids+infesting+conifer+bark+in+Europe+in+relation+to+the+biological+control+of+Dendroctonus.+spp+in+Canada.">The natural enemies of scolytids infesting conifer bark in Europe in relation to the biological control of Dendroctonus. spp in Canada.</a> Biocontrol News Information. 4, 305-328 (1983).</li><li>Nagel, W. P., Fitzgerald, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medetera+aldrichii.+larval+feeding+behavior+and+prey+consumption+[Dipt.:+Dolichopodidae].">Medetera aldrichii. larval feeding behavior and prey consumption [Dipt.: Dolichopodidae].</a> Entomophaga. 20, 121-127 (1975).</li><li>Paine, T., Raffa, K., Harrington, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+among+scolytid+bark+beetles,+their+associated+fungi,+and+live+host+conifers.">Interactions among scolytid bark beetles, their associated fungi, and live host conifers.</a> Annu Rev Entomol. 42, 179-206 (1997).</li><li>Reeve, J. D., Coulson, R. N., Klepzig, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predators+of+the+southern+pine+beetle.">Predators of the southern pine beetle.</a> Southern Pine Beetle II. , 153-160 .</li><li>Reid, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behavior+of+the+mountain+pine+beetle,+Dendroctonus+monticolae.+Hopkins,+during+mating,+egg+laying+and+gallery+construction.">The behavior of the mountain pine beetle, Dendroctonus monticolae. Hopkins, during mating, egg laying and gallery construction.</a> Can Entomol. 90, 505-509 (1958).</li><li>Taylor, A. D., Hayes, J. L., Roton, L., Moser, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phloem+sandwich+allowing+attack+and+colonization+by+bark+beetles+(Coleoptera:+Scolytidae)+and+associates.">A phloem sandwich allowing attack and colonization by bark beetles (Coleoptera: Scolytidae) and associates.</a> J. Entomol. Soc. Amer. 27, 101-116 (1992).</li><li>Yu, C. C., Tsao, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gallery+construction+and+sexual+behavior+in+the+southern+pine+beetle,+Dendroctonus+frontalis.+Zimm.+(Coleoptera:+Scolytidae).">Gallery construction and sexual behavior in the southern pine beetle, Dendroctonus frontalis. Zimm. (Coleoptera: Scolytidae).</a> Georgia Entomology Society. 2, 95-98 (1967).</li><li>Yturralde, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Acoustic+Ecology+of+Bark+Beetles+and+Bed+Bugs.">The Acoustic Ecology of Bark Beetles and Bed Bugs.</a> PhD. Dissertation. , 323 (2013).</li><li>Wermelinger, B., Seifert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+temperature+dependent+development+of+the+spruce+bark+beetle+Ips+typographus+(L.)+(Col.,+Scolytidae).">Analysis of the temperature dependent development of the spruce bark beetle Ips typographus (L.) (Col., Scolytidae).</a> Journal of Applied Entomology. 122, 185-191 (1998).</li><li>Whitney, H. S., Mitton, J. B., Sturgeon, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+between+bark+beetles+and+symbiotic+organisms.">Relationships between bark beetles and symbiotic organisms.</a> Bark Beetles in North American Conifers. , 183-211 (1982).</li><li>Wood, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bark+and+ambrosia+beetles+of+North+and+Central+America+(Coleoptera:+Scolytidae),+a+taxonomic+monograph.">The bark and ambrosia beetles of North and Central America (Coleoptera: Scolytidae), a taxonomic monograph.</a> , 1359 (1982).</li></ol>

No conflicts of interest declared

The phloem sandwich allows for the introduction and observation of arthropods, microbes, and other small organisms that inhabit phloem tissues.1,7,8,9,17,18 This technique has resulted in new discoveries and a better understanding of behaviors, life-history traits, development, and interactions of organisms within tree phloem.1,5,10 The sandwich protocol described here is a hybrid of past designs, and will provide an economical sandwich that is easily constructed with minimal equipment and materials. The glass, acrylic, or polycarbonate pieces can be reused, and the only consumable materials are the Parafilm and phloem.
Although all steps in the protocol are important, certain steps should be followed strictly to ensure the highest degree of success. First, a tree should be located that has a portion of the bole free, or relatively free, of branches. Trees that have many branches are difficult to shave and will yield few viable phloem pieces, as every branch must be cut around when removing the phloem. Next, it is important to minimize air exposure of the phloem. We quickly place each phloem piece in a bag upon removal. Once three to six pieces are removed, they are transferred into a vacuum-sealed bag; we use a power inverter to run the sealer off a vehicle battery in the field. Last, sanitizing the acrylic pieces and general cleanliness when executing this protocol will reduce fungal growth in the sandwich. This is especially important for extended observations.
As mentioned above, there are limitations of the phloem sandwich apparatus. Since the phloem layer is thin, only small organisms that typically remain in the phloem can be introduced into the sandwich. Larger insects such as wood borers (i.e., Buprestid, Cerambycid species) can be introduced and observed for the early stages of their lifecycle. This timeframe is usually limited to two to three weeks; after this point the larvae require xylem wood to bore into for pupation. In contrast, for bark beetles, especially those in the genus Ips, a full lifecycle can be observed&#8212;including mating, egg hatching, feeding, pupating, and eclosion into an adult beetle. At this point, the viability of the sandwich is usually exhausted due to desiccation and fungal growth.28 Additionally, this apparatus does not allow insects to freely and naturally colonize or exit the phloem sandwich.27
Our protocol is flexible in terms of size, shape, and type of phloem used. Short studies require less phloem material and sandwich size can be scaled accordingly. Many coniferous species have been used as phloem donors in a phloem sandwich (e.g., ponderosa pine12, Douglas-fir2, spruce29, loblolly pine27, longleaf pine27). Materials in the sandwich can also be altered; for example, glass plates can be used instead of acrylic and epoxy or tape instead of Parafilm.
The most difficult part of this protocol is the phloem removal process. Trees next to each other may differ in how difficult their phloem is to remove. When a tree has difficult phloem, patience is critical. In these cases, carefully run a knife between the hard xylem and the spongy phloem. This process feels literally like skinning the tree.
After mastering the basic sandwich technique, alterations to the protocol can help fit specific needs. For example, by honing the techniques required to remove phloem, larger pieces can be removed and used to create larger sandwiches. Also, alterations can be made to accommodate specific instruments, e.g., additional holes to monitor chemical emissions (Fig. 3A), or record or playback sounds (Fig. 3B). Modifications can be made to allow for semi-permanent preservation of the phloem and its organisms, or for temporary observations of organisms that can later be let free.

The phloem and bark tissues of conifers are host to thousands of organisms. Phloeophagy, the feeding on phloem tissues of the inner bark, is a habit usually associated with bark beetles, woodborers, and several other invertebrate and microbial taxa that live within trees.23 Bark beetles (Coleoptera: Curculionidae) develop and live within phloem except for short periods when adults seek new host trees.31 Bark beetles have been extensively studied due to their economic impacts on trees18,19, but direct behavioral observations of the insects within tree materials have been limited.4 Furthermore, the galleries constructed by bark beetles become habitat for a myriad of species.11 Vast numbers of fungi30, bacteria3, mites10,21, and nematodes16,19 along with other predatory and parasitic arthropods22,24 inhabit the phloem material. The techniques provided here allow for direct observation of bark beetles, mites, and wood borers that typically live in subcortical environments. Slight alterations to the protocol can be made to study fungi and bacteria.
Bark beetles and associated organisms within tree tissues have been studied using a &#8220;phloem sandwich.&#8221; Early use of this technique can be found in the literature dating back to 1933, when it was used to observe the larval instars of Douglas-fir beetle (Dendroctonus pseudotsugae).2 The phloem sandwich has gone through many derivations as different materials became available. Originally, this device consisted of a piece of phloem placed between two plates of glass, pressed together by elastic bands.2 Later, clamps, tape, glue, plastic, and other materials have been used in the construction of the sandwich.13,14,15,17,26,28 The protocol described here offers improvements over some of the past designs. For example, in the past, test species were entered into the side of the sandwich, in between the plates of glass or plastic. This restricted the construction of galleries to one direction. The use of entry holes in the top plate allows greater freedom for test species to initiate natural gallery construction. Another advantage of the presented protocol is its simplified design, which can be easily constructed with few tools. The use of the phloem sandwich has allowed for direct observations of feeding behavior, reproduction, development, and interactions of organisms that would otherwise not have been possible.1,5, 22 This method is also an excellent tool for K-12 education and science programs and displays.
There are several subtleties in the creation of a phloem sandwich that are difficult to interpret from or not reported in manuscripts. We believe that a visual (i.e., video) description of the production of a phloem sandwich is needed and would be of value to scientists and educators interested in studying phloeophagous organisms. Our protocol provides a simple and inexpensive way to observe arthropods, microbes, and other organisms that inhabit phloem tissues.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Draw blade</td>
			<td>Big Horn Brand&#160;</td>
			<td>20265</td>
			<td>11&#8221; blade</td>
		</tr>
		<tr>
			<td>Fillet Knife</td>
			<td>American Angler</td>
			<td>30530</td>
			<td>9&#8221; blade</td>
		</tr>
		<tr>
			<td>Polycarbonate</td>
			<td>Nexan</td>
			<td>GE-33</td>
			<td>0.093 in. thickness</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Fisher Scientific</td>
			<td>S37441</td>
			<td>2&#8221; wide</td>
		</tr>
		<tr>
			<td>Clamps</td>
			<td>Pony Jaw Opening</td>
			<td>3201-HT-K</td>
			<td>4&#8221; x 1&#8221;</td>
		</tr>
		<tr>
			<td>Vacuum Sealer&#160;</td>
			<td>FoodSaver</td>
			<td>V2840,&#160;</td>
			<td>VacLoc vacuum&#160;</td>
		</tr>
		<tr>
			<td>and bags</td>
			<td></td>
			<td>FSFSBF0742-015</td>
			<td>bags in rolls</td>
		</tr>
	</tbody>
</table>

technique for studying arthropod and microbial communities within tree tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a &#8216;phloem sandwich&#8217; and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.

The protocol described above will enable a researcher to observe organisms that live in the cryptic environment underneath the bark of a tree. To illustrate the use of this technique we describe a representative study from our lab that uses this technique.12 In this experiment, phloem sandwiches were used to observe the effects of acoustic treatments on reproductive output, tunneling distance, and survival of bark beetles (Fig. 2E and 2F). Direct observation of beetles, made possible by the use of a phloem sandwich, revealed several interesting findings. First, we recorded daily tunneling distances by marking the beetles&#8217; location on the acrylic with a marker every 24h. This process revealed significant differences across sound treatments that would have been hidden without the phloem sandwich. Next, we observed a reduction in egg production with particular sound treatments. The mobile nature of the phloem sandwich allowed for egg observations to be completed under a dissecting microscope without disturbing the beetles. During these observations, we captured beetle egg laying behavior with a high-definition camera attached to the microscope. Other notable observations included the killing of mates and the initiation of flight associated with particular acoustic treatments. The phloem sandwich assay was crucial to our study of bark beetle responses to acoustic treatments. These discoveries, which would be impossible to observe underneath the bark of a tree, are valuable contributions to developing management options for combatting bark beetle outbreaks.
<img alt="Figure 1" src="/files/ftp_upload/50793/50793fig1highres.jpg" /> 
Figure 1. A) Tools needed to remove bark from tree. Items 1 and 2 are draw blades used to scrape bark off of the tree. Items 3 (hatchet) and 4 (pull saw) are useful to remove branches near shaving area. B) Use of draw blade to remove bark from tree. Notice the light color of the phloem underneath the reddish bark.
<img alt="Figure 2" src="/files/ftp_upload/50793/50793fig2highres.jpg" /> 
Figure 2. A) Removal of phloem after bark was scraped off of tree. B) Fresh phloem stored in vacuum sealed bag. C) Phloem sandwich with clamps holding the acrylic pieces together and Parafilm around edges to prevent contamination and desiccation of phloem. D) Bark beetle near hole drilled into acrylic . E) Using microscope to observe phloem sandwich. F) Video display of bark beetles within phloem sandwich.
<img alt="Figure 3" src="/files/ftp_upload/50793/50793fig3highres.jpg" /> 
Figure 3. A) Pheromone acquisition from beetles in phloem sandwich. Air is being pulled through tubes containing Super-Q absorbent. B) Recording and playback of sound via a piezo-electric transducer (center of picture) to mountain pine beetles within phloem sandwich.

Watch this Scientific Journal Video about Technique for Studying Arthropod and Microbial Communities within Tree Tissues at JoVE.com

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

technique-for-studying-arthropod-microbial-communities-within-tree

Nanyang Technological University

Research

JoVE Journal

Environment

10.3K Views.  Northern Arizona University. We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues. 

Video: Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep), peak current density (jp), formal potential (Ef) and peak separation (&#916;Ep). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.

Chronoamperometric growth of anodic electroactive microbial biofilms in a fed-batch reactor using a three-electrode setup, controlled by a potentiostat, is demonstrated. The extracellular electron transfer is characterized using cyclic voltammetry in the presence of the electron donor and in the absence of the electron donor. Fundamental data analysis is demonstrated.

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup ...

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

A Technical Perspective in Modern Tree-ring Research - How to Overcome Dendroecological and Wood Anatomical Challenges

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to environmental changes and to finally reconstruct such changes. This is done by analyzing growth variations back in time and correlating various plant-specific parameters to (for example) temperature records. Integrating wood anatomical parameters in these analyses would strengthen reconstructions, even down to intra-annual resolution. We therefore present a protocol on how to sample, prepare, and analyze wooden specimen for common macroscopic analyses, but also for subsequent microscopic analyses. Furthermore we introduce a potential solution for analyzing digital images generated from common small and large specimens to support time-series analyses. The protocol presents the basic steps as they currently can be used. Beyond this, there is an ongoing need for the improvement of existing techniques, and development of new techniques, to record and quantify past and ongoing environmental processes. Traditional wood anatomical research needs to be expanded to include ecological information to this field of research. This would support dendro-scientists who intend to analyze new parameters and develop new methodologies to understand the short and long term effects of specific environmental factors on the anatomy of woody plants.

Here we present a protocol outlining how to sample wooden specimens for the overall assessment of their growth structures. Macro- and microscopic preparation and visualization techniques necessary to generate well-replicated and highly resolved wood anatomical and dendroecological dataset, are described are described.

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to ...

Watershed Planning within a Quantitative Scenario Analysis Framework

There is a critical need for tools and methodologies capable of managing aquatic systems within heavily impacted watersheds. Current efforts often fall short as a result of an inability to quantify and predict complex cumulative effects of current and future land use scenarios at relevant spatial scales. The goal of this manuscript is to provide methods for conducting a targeted watershed assessment that enables resource managers to produce landscape-based cumulative effects models for use within a scenario analysis management framework. Sites are first selected for inclusion within the watershed assessment by identifying sites that fall along independent gradients and combinations of known stressors. Field and laboratory techniques are then used to obtain data on the physical, chemical, and biological effects of multiple land use activities. Multiple linear regression analysis is then used to produce landscape-based cumulative effects models for predicting aquatic conditions. Lastly, methods for incorporating cumulative effects models within a scenario analysis framework for guiding management and regulatory decisions (e.g., permitting and mitigation) within actively developing watersheds are discussed and demonstrated for 2 sub-watersheds within the mountaintop mining region of central Appalachia. The watershed assessment and management approach provided herein enables resource managers to facilitate economic and development activity while protecting aquatic resources and producing opportunity for net ecological benefits through targeted remediation.

There is a critical need for tools and methodologies capable of managing aquatic systems in the face of uncertain future conditions. We provide methods for conducting a targeted watershed assessment that enables resource managers to produce landscape-based cumulative effects models for use within a scenario analysis management framework.

There is a critical need for tools and methodologies capable of managing aquatic systems within heavily impacted watersheds. Current efforts often fall ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...

Characterizing Microbiome Dynamics &#8211; Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical applications driven by microorganisms. Next generation sequencing methods are widely utilized to resolve microbiomes, but they are generally resource and time intensive and deliver mostly qualitative information. Flow cytometric microbiome analysis does not suffer from those disadvantages and can provide relative subcommunity abundances and absolute cell numbers at-line. Although it does not deliver direct phylogenetic information, it can enhance the analysis depth and resolution of sequencing approaches. In sharp contrast to medical applications in both research and routine settings, flow cytometry is still not widely used for microbiome analysis. Missing information on sample preparation and data analysis pipelines may create an entry barrier for the researchers facing microbiome analysis challenges that would often be textbook flow cytometry applications. Here, we present three comprehensive workflows for pure cultures, complex communities in clear medium and complex communities in challenging matrices, respectively. We describe individual sampling and fixation procedures and optimized staining protocols for the respective sample sets. We elaborate the cytometric analysis with a complex research centered and an application focused bench top device, describe the cell sorting procedure and suggest data analysis packages. We furthermore propose important experimental controls and apply the presented workflows to the respective sample sets.

Flow cytometric analysis has proven valuable for investigating pure cultures and monitoring microbial community dynamics. We present three comprehensive workflows, from sampling to data analysis, for pure cultures and complex communities in clear medium as well as in challenging matrices, respectively.

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical ...

Automated 3D Optical Coherence Tomography to Elucidate Biofilm Morphogenesis Over Large Spatial Scales

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm morphogenesis, that is the structural diversification of biofilms during community assembly, represents a remarkable challenge across spatial and temporal scales. Here, we present an automated biofilm imaging system based on optical coherence tomography (OCT). OCT is an emerging imaging technique in biofilm research. However, the amount of data that currently can be acquired and processed hampers the statistical inference of large scale patterns in biofilm morphology. The automated OCT imaging system allows covering large spatial and extended temporal scales of biofilm growth. It combines a commercially available OCT system with a robotic positioning platform and a suite of software solutions to control the positioning of the OCT scanning probe, as well as the acquisition and processing of 3D biofilm imaging datasets. This setup allows the in situ and non-invasive automated&#160;monitoring of biofilm development and may be further developed to couple OCT imaging with macrophotography and microsensor profiling.

Microbial biofilms form complex architectures at interphases and develop into highly scale-dependent spatial patterns. Here, we introduce an experimental system (hard- and software) for the automated acquisition of 3D optical coherence tomography (OCT) datasets. This toolset allows the non-invasive and multi-scale characterization of biofilm morphogenesis in space and time.