The authors are grateful to the Forest Service of Canton Ticino and the Federal Office for the Environment FOEN for partially funding this study.

1. Tree Selection and Assessment Design
<ol>
	<li>If possible, identify the epidemic stage of the study area by determining the arrival years of Dryocosmus kuriphilus and Torymus sinensis and the T. sinensis parasitization rate by using reliable sources (e.g., scientific publications, forest services, chestnut grove managers&#39; knowledge).
	<ol>
		<li>If no reliable sources are available, identify the four main epidemic stages (Early, Peak, Recovery, Biocontrol) by computing the T. sinensis parasitization rate combined with the field observations described below.
		<ol>
			<li>Identify the stage as early epidemic when tree crowns show neither significant damage nor crown transparency, current year galls are rare, and T. sinensis parasitization is very low or negligible.</li>
			<li>Identify epidemic peak when tree crowns display a high degree of transparency, although dead branches are rare, and current year galls are abundant.</li>
			<li>Identify prolonged epidemic peak when current and previous years&#39; galls are abundant (up to three years previous) and T. sinensis parasitization is still very low or absent. Tree crowns still display high transparency and additional damage is represented by the first evidence of dead branches in the crown.</li>
			<li>Identify early recovery stage when T. sinensis parasitization rate becomes greater than 75%10. Damage is still high but the number of current year galls decreases and some branches produce gall-free shoots, especially from the apical bud.</li>
			<li>Identify recovery stage when T. sinensis parasitization rate is permanently greater than 75%, current year galls are rare and usually limited to single trees only and most branches produce gall-free shoots. Past years&#39; galls on older branches and dead branches due to D. kuriphilus attacks are still visible.</li>
			<li>Identify fully recovered stage when damage and galls (past and current year) are rare or absent and crowns are fully recovered. In severely damaged trees, some vestiges (e.g., dead shoots or rotten past years&#39; galls) of prior D. kuriphilus attacks can still be present inside the crown. 
			NOTE: Supplemental File 1 shows exemplary tree crown pictures for each epidemic stage.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Observe chestnut trees in the whole area to visually estimate damage variability among and within trees. Damage variability is usually low during the early epidemic and the recovery stages (crowns are basically healthy) as well as during the epidemic peak (whole crowns are full of galls). In contrast, variability tends to be high in the intermediate epidemic stages, when dead shoots due to past D. kuriphilus attacks are still present.</li>
	<li>Based on 1.1 and 1.2, determine the number of trees to analyze. Unfortunately, it is not possible or suitable to give a specific rule regarding sample size, which may vary according to the specific epidemic situation in the field and/or the research objectives. Based on our 10 years of experience, for a 10-hectare site we advise the following (also see Table 1 for details):
	<ol>
		<li>Sample at least ten trees per site, regardless of the epidemic stage. Although during the early epidemic and recovered stage three trees would be enough, increasing the sample size to ten will give more statistical power to the results.</li>
		<li>During the early epidemic and recovered stage, sample one branch per tree.</li>
		<li>During the epidemic peak, sample one branch per tree if galls are evenly distributed within the tree crown, or two branches per tree if you notice that some crown parts have been attacked more severely.</li>
		<li>During the other epidemic stages, increase the number of branches to two (for trees that are recovering well) or three (for more damaged trees) based on the variability of crown damage of every tree.</li>
	</ol>
	</li>
</ol>
2. Data Collection in the Field
<ol>
	<li>Prepare the appropriate equipment including a clipboard, a camping chair, secateurs, a telescopic tree pruner, a 30 m measuring tape, and tree climbing equipment if the top crown above 8 m&#160;requires analysis.</li>
	<li>Select the most representative branches trying to proportionally cover branch diversity within the tree crown (healthy trees usually have similar branches while damaged trees may have branches with different degrees of damage). For example, if you choose to collect three branches per tree, collect the most damaged branch, the healthiest and an intermediate one.</li>
	<li>Whenever possible, select architectural branches only, while avoiding reiterations (trunk suckers or reiterations sensu Hall&#233;)16.</li>
	<li>Ensure branches are at least 50 cm&#160;in length and have at least 10 shoots.</li>
	<li>Attach the beginning of the measuring tape near the blade of the telescopic tree pruner in order to measure the height above ground of the branch at the cutting point.</li>
	<li>Cut the branch with the telescopic pruner, record its cutting height, its aspect, its type (architectural or reiteration) and refine the branch selection with secateurs in order to keep only the part for analysis.</li>
	<li>Assign a unique ID to the branch and record its age, its maximum length (from the first branching point to the apex) for general information.</li>
	<li>Take a quick look at the whole branch to obtain a first impression of its history and present status (heavily attacked or not) and identify all the elements and features important for the calculation of the DCI with the help of Figure 2 and Figure 3.</li>
</ol>
3. Branch Feature Definition
The following definitions are partially or totally reproduced from Gehring et al.&#160;20185, with the permission of Springer-Verlag Berlin Heidelberg 2017.
<ol>
	<li>Define a Sprout (on a shoot) as a freshly formed sprout that has grown during the current vegetative season from a developed bud on a shoot.</li>
	<li>Define a Shoot as a sprout from the previous vegetative season with respect to the sampling date (e.g., sampling season = 2017, shoot = sprout that grew in 2016). Can be dead or alive.</li>
	<li>Define a Dead shoot (Sd) as a dead shoot after D. kuriphilus attack or due to natural death.</li>
	<li>Define an Alive shoot (As) as a living shoot, not to be confused with a reactivated dormant bud.</li>
	<li>Define a Reactivated dormant bud (Bdor) as a freshly formed sprout that has grown during the current vegetative season from a dormant bud on a multiyear branch part that is older than the shoot.</li>
	<li>Define a Gall on shoot (Gons) as a gall developed at the base or along the axis of a sprout. Technically, these should be called &#34;galls on sprouts&#34; but for the purpose of consistency with existing literature, we refer to them as &#34;galls on shoots&#34;. 
	NOTE: Figure 2 and Figure 3 show examples of selected branch features. More detailed and complete descriptions (which are beyond the scope of this paper) may be found in Gehring et al.&#160;20185 and Maltoni et al.&#160;20124.</li>
</ol>
4. Branch Analysis
<ol>
	<li>Count and record all the living shoots (alive shoots).</li>
	<li>Count and record all the dead shoots.</li>
	<li>Count and record all the reactivated dormant buds.</li>
	<li>Count and record all the galls on shoots. 
	NOTE: Supplemental File 2 shows an example of a field sampling form and Supplemental File 3 shows the sampling form filled out.</li>
</ol>
5. Calculation of the Damage Composite Index
<ol>
	<li>Calculate the proportion of dead shoots (Sd) from the number of dead shoots divided by the total number of shoots (dead shoots + alive shoots).</li>
	<li>Calculate the proportion of reactivated dormant buds (BdoR) from the number of reactivated dormant buds divided by the total number of living shoots (BdoR + alive shoots).</li>
	<li>Calculate the average number of galls on shoots (Gons) from the number of galls on shoots divided by the number of living shoots (BdoR + alive shoots).</li>
	<li>Calculate the DCI using the formula DCI = (Sd * 0.479 + BdoR * 0.525 + Gons * 0.120) * 100.</li>
	<li>Use Table 2 to evaluate the damage severity. 
	Note: An R script with the DCI function is available in Supplemental Coding File 1. Its description is found in Supplemental File 4.</li>
</ol>

<ol>
	<li>Stone, G. N., Sch&#246;nrogge, K., Rachel, J., Bellido, D., Pujade-villar, 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+population+biology+of+oak+gall+wasps+(Hymenoptera:+Cynipidae).">The population biology of oak gall wasps (Hymenoptera: Cynipidae).</a> Annual Review of Entomology. (47), 633-668 (2002).</li><li>Abe, Y., Melika, G., Stone, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diversity+and+phylogeography+of+cynipid+gallwasps+(Hymenoptera:+Cynipidae)+of+the+Oriental+and+eastern+Palearctic+regions,+and+their+associated+communities.">The diversity and phylogeography of cynipid gallwasps (Hymenoptera: Cynipidae) of the Oriental and eastern Palearctic regions, and their associated communities.</a> Oriental Insects. 41 (1), 169-212 (2007).</li><li>Aebi, A., Schoenenberger, N., Bigler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+use+of+Torymus+sinensis+against+the+chestnut+gall+wasp+Dryocosmus+kuriphilus+in+the+Canton+Ticino,+Switzerland.">Evaluating the use of Torymus sinensis against the chestnut gall wasp Dryocosmus kuriphilus in the Canton Ticino, Switzerland.</a> Agroscope Reckenholz-T&#228;nikon Report. , (2011).</li><li>Maltoni, A., Mariotti, B., Tani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Case+study+of+a+new+method+for+the+classification+and+analysis+of+Dryocosmus+kuriphilus+Yasumatsu+damage+to+young+chestnut+sprouts.">Case study of a new method for the classification and analysis of Dryocosmus kuriphilus Yasumatsu damage to young chestnut sprouts.</a> IForest. 5 (1), 50-59 (2012).</li><li>Gehring, E., Bellosi, B., Quacchia, A., Conedera, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+impact+of+Dryocosmus+kuriphilius+on+the+chestnut+tree+branch+architecture+matters.">Assessing the impact of Dryocosmus kuriphilius on the chestnut tree branch architecture matters.</a> Journal of Pest Science. 91 (1), 189-202 (2018).</li><li>Kato, K., Hijii, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+gall+formation+by+Dryocosmus+kuriphilus+Yasumatsu+(Hym+.,+Cynipidae+)+on+the+growth+of+chestnut+trees.">Effects of gall formation by Dryocosmus kuriphilus Yasumatsu (Hym ., Cynipidae ) on the growth of chestnut trees.</a> Journal of Applied Entomology. 121 (1-5), 9-15 (1997).</li><li>Meyer, J. B., Gallien, L., Prospero, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+two+invasive+organisms+on+the+European+chestnut:+Does+the+chestnut+blight+fungus+benefit+from+the+presence+of+the+gall+wasp?.">Interaction between two invasive organisms on the European chestnut: Does the chestnut blight fungus benefit from the presence of the gall wasp?.</a> FEMS Microbiology Ecology. 91 (11), 1-10 (2015).</li><li>Turchetti, T., Addario, E., Maresi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+chestnut+gall+wasp+and+blight:+a+new+criticality+for+chestnut.">Interactions between chestnut gall wasp and blight: a new criticality for chestnut.</a> Forest@ - Rivista di Selvicoltura ed Ecologia Forestale. 7 (1), 252-258 (2010).</li><li>Moriya, S., Shiga, M., Adachi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+biological+control+of+the+chestnut+gall+wasp+in+Japan.">Classical biological control of the chestnut gall wasp in Japan.</a> , 407-415 (2003).</li><li>Quacchia, A., Moriya, S., Bosio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+Torymus+sinensis+in+the+Biological+Control+of+Dryocosmus+kuriphilus+in+Italy.">Effectiveness of Torymus sinensis in the Biological Control of Dryocosmus kuriphilus in Italy.</a> Acta Horticulturae. 1043, 199-204 (2014).</li><li>Kotobuki, K., Mori, K., Sato, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2+methods+to+estimate+the+tree+damage+by+chestnut+gall+wasp+Dryocosmus-kuriphilus.">2 methods to estimate the tree damage by chestnut gall wasp Dryocosmus-kuriphilus.</a> Bulletin of the fruit tree research station A (Yatabe). 2 (12), 29-36 (1985).</li><li>Sartor, C., Dini, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+the+Asian+wasp+Dryocosmus+kuriphilus+(Yasumatsu)+on+cultivated+chestnut:+Yield+loss+and+cultivar+susceptibility.">Impact of the Asian wasp Dryocosmus kuriphilus (Yasumatsu) on cultivated chestnut: Yield loss and cultivar susceptibility.</a> Scientia Horticulturae. 1997, 454-460 (2015).</li><li>Johnstone, D., Moore, G., Tausz, M., Nicolas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement+of+plant+vitality+in+landscape+trees.">The measurement of plant vitality in landscape trees.</a> Arboricultural Journal: The International Journal of Urban Forestry. 35 (1), 18-27 (2013).</li><li>Guyot, V., Castagneyrol, B., Deconchat, M., Selvi, F., Bussotti, F., Jactel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tree+diversity+limits+the+impact+of+an+invasive+forest+pest.">Tree diversity limits the impact of an invasive forest pest.</a> Plos One. , (2015).</li><li>Gehring, E., Bosio, G., Quacchia, A., Conedera, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adapting+sampling+effort+to+assess+the+population+establishment+of+Torymus+sinensis,+the+biocontrol+agent+of+the+chestnut+gallwasp.">Adapting sampling effort to assess the population establishment of Torymus sinensis, the biocontrol agent of the chestnut gallwasp.</a> International Journal of Pest Management. , (2017).</li><li>Hall&#233;, F., Oldeman, R. A. A., Tomlinson, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Formation of Trees and Forests. An architectural analysis. , (1978).</li><li>Gyoutoku, Y., Uemura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecology+and+biological+control+of+the+chestnut+gall+wasp,+Dryocosmus+kuriphilus+Yasumatsu+(Hymenoptera:+Cynipidae).+1.+Damage+and+parasitization+in+Kumamoto+Prefecture.">Ecology and biological control of the chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae). 1. Damage and parasitization in Kumamoto Prefecture.</a> Proceedings of the Association for Plant Protection of Kyushu (Japan). 31, 213-215 (1985).</li><li>M&#252;ller, E., Stierlin, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sanasilva+Kronenbilder+mit+Nadel-+und+Blattverlustprozenten.">Sanasilva Kronenbilder mit Nadel- und Blattverlustprozenten.</a> Eidgeno&#776;ssische Forschungsanstalt fu&#776;r Wald, Schnee und Landschaft: Birmensdorf. , (1990).</li></ol>

The authors have nothing to disclose.

Dryocosmus kuriphilus lay eggs in chestnut tree buds, inducing the formation of galls in spring. Repeated and uncontrolled D. kuriphilus attacks cause, in addition to gall formation, general branch corruption, including the death of many shoots and a significant loss in green photosynthetic leaf area5. Trees usually react by attempting to produce substitutive shoots via the activation of dormant buds. For this reason, especially during the epidemic peak and the recovery stage, classic MAID (based on galls abundance only) tend to underestimate the real damage caused by D. kuriphilus while the DCI, which is based not only on gall abundance but also on dead shoots and reactivated dormant buds, better reflects real damage severity and the degree of branch architecture alteration. In fact, MAID focus more on the population level of D. kuriphilus rather than on the degree of damage to trees. For example, when the branch damage is low and the presence of galls is insignificant during the early epidemic and the final, recovered stage, both MAID and DCI show very low values. However, during the epidemic peak or when exposed to several years of D.kuriphilus attack, a severely damaged branch may display many dead parts but only a few galls, or none at all. Using the criteria for each index, this would result in high DCI values (severe damage) but low MAID values (low damage).
It is thus important to understand the meaning and related damage degree indicated by each index in order to select the most suitable pest assessment approach according to the desired purpose. We therefore suggest evaluating the overall impact of the pest by using the DCI for the assessment of branch architecture damage, especially during the epidemic peak and the recovery stage. To ensure thorough pest assessment, classic MAID may be used to evaluate pest population levels (we suggest Gehring et al.&#160;201715 for advice on how to adapt the sampling effort) whereas DCI may be used for a detailed assessment of branch architecture as well as for the entire tree crown. For a simple general assessment of crown transparency, in contrast, a tree assessment using the SANASILVA crown transparency index18 may be the most suitable in terms of effort-benefit balance.
Once the operator becomes familiar with the main branch structures and features needed, applying the DCI is quite simple and relatively fast. However, in case of highly damaged branches due to repeated D. kuriphilus attacks, it could be difficult to properly assess the number of dead shoots because of the presence of older dead branches. It is thus important to try to reconstruct the branch history and to evaluate the degree of rot in the dead branches and shoots. Usually, dead shoots are not rotten or are less so compared to dead branches.
At the tree level, the major difficulty when evaluating the average branch damage of a large tree with heterogeneous crown damage (e.g., a tree displaying healthy, damaged, and dead branches) regards the quantity of branches for analysis and the tree climbing skills and effort needed to reach the top of the crown. Moreover, since the method is destructive, evaluating a damaged tree will inevitably inflict a temporary additional loss of green biomass. It is therefore important to only select the smallest number of most representative branches in order to avoid excessive cutting.
Deciding on the correct sample size can sometimes be tricky. Based on our experience we note that in both the early epidemic and the fully recovered stage, damage on trees is absent and, consequently, its variability is very low. Consequently, sampling 10 trees per site and 1 branch per tree already gives a fair estimate of the damage. In contrast, during the epidemic peak and the recovery stage, trees show different damage levels. A good balance between sampling effort, data accuracy, and tree damage infliction can be reached by collecting 3 branches per tree for a total of 10 trees per site. Please note that this advice is based on our experience and research needs. Others are free to increase or decrease sample sizes according to their specific situation and assessment goals.
Finally, it is possible that at the beginning of the recovered phase, when almost no galls or dead shoots are visible on branches any longer, the DCI (and MAID) underestimate the residual damage present on the branches. This residual damage is represented by the possible lack of lateral shoots and dormant buds that occur especially in severely damaged trees that have suffered from repeated D. kuriphilus attacks over the years5. The lack of lateral shoots implies that a part of the leaf area is still missing, whereas the lack of dormant buds indicates that the tree has still not fully recovered its reserves.
Future or other applications of the DCI are difficult to imagine because it is species-specific and the constants applied in its calculation are calibrated to the chestnut tree5. Nevertheless, the methodology used to create it could be adapted and implemented to any other tree species and related pest.

The chestnut gallwasp Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) is the most significant global insect pest of the genus Castanea1,2,3. Through its repeated galling activity, it prevents and inhibits normal shoot development4,5, causing a progressive reduction of leaf area and a consequent loss of tree green biomass and vigour5,6, dormant bud reactivation5 and an increase in gallwasp post-emergence branch mortality7,8.
The European experience of the gallwasp epidemic shows that uncontrolled and repeated gallwasp attacks may induce a high level of crown corruption in Sweet chestnut (Castanea sativa Mill.). This can result in crown leaf area losses of up to 70% that are neither compensated for by substitutive foliage produced by the activation of dormant buds nor by building second flushes during the same vegetation period5.
The only successful method to reduce the pest population and allow chestnut trees to recover is biological control through its natural antagonist the parasitoid Torymus sinensis Kamijo (Hymenoptera: Torymidae)9,10. Once biological control through its natural enemy is achieved, the chestnut trees start to produce new healthy sprouts. If tree damage level is very high, this may occur starting from the terminal bud only, due to the fact that it is usually infestation-free because of its formation after gallwasp oviposition activity4. This implies a long recovery process before the whole tree crown is re-established5.
In order to check the positive reaction of chestnut trees after biological control by Torymus sinensis is reached, and to verify the need for sylvicultural (pruning, thinning) intervention, forest managers and chestnut growers need a method for quick and reliable assessment of damage level and related branch architecture and leaf area evolution throughout the gallwasp epidemic from the initial infestation phase by the pest to recovery after biological control by its antagonist. Several methods for assessing gallwasp infestation degree (MAID) have been developed and used worldwide to date, such as measuring the proportion of attacked buds11 or the average number of galls per bud12. MAID do not directly measure green biomass (e.g., leaf area), reserve structures such as dormant buds, reaction structures (e.g., reactivated dormant buds and second flushes), or previous year damage (e.g., dead shoots) as major proxies of current tree vitality and vigour6,13,14. Moreover, most MAID are only based on the number of galls found on tree branches and underestimate real branch damage, especially during the peak of the pest epidemic (Figure 1).
In this paper, we describe the damage composite index (DCI) approach proposed by Gehring et al.&#160;20185 that considers proxies of green biomass, reserves such as dormant bud, and tree reactions (dormant bud reactivation and second flushes), enabling a realistic, reliable, and reasonably rapid assessment of damage through all stages of an epidemic, especially when combined with the assessment effort optimization proposed by Gehring et al.&#160;201715.
In particular, the objectives of this paper are 1) to give a detailed description of the field protocol, including the relevant branch features to be assessed, 2) to present the damage composite index formula, and 3) to propose an improved severity scale conversion for the DCI.

<table><tbody><tr><td>Name of Material/ Equipment</td><td>Company</td><td>Catalog Number</td><td>Comments</td></tr><tr><td>Clipboard</td><td>Any brand</td><td></td><td></td></tr><tr><td>Camping chair&lt;br/&gt; (Foldable and lightweight chair)</td><td>Any brand</td><td></td><td>Companies: Kelty, Campz, Half-Ton.</td></tr><tr><td>Felco 9 secateurs&lt;br/&gt; (One-hand pruning shear)</td><td>Felco</td><td></td><td>Other companies: Bahco.</td></tr><tr><td>AP-5M-Aluminium Pole&lt;br/&gt; (Telescopic tree pruner pole)</td><td>Bahco</td><td>8152079</td><td>Other companies: Spear &amp;amp; Jackson, Kingfisher, Hortex, Fiskars.</td></tr><tr><td>P34-37 top pruner&lt;br/&gt; (Telescopic tree pruner head)</td><td>Bahco</td><td>8002787</td><td></td></tr><tr><td>100 ft Fiberglass Long Tape&lt;br/&gt; (30 m measuring tape)</td><td>Stanley</td><td>34-790</td><td>Other companies: Tjima, Freemans, Astor, Lux.</td></tr><tr><td>Parallel 10.5mm&lt;br/&gt; (Low stretch kernmantel rope, flexible and lightweight for rope access)</td><td>Petzl</td><td>R077AA03</td><td>Basic equipment for tree climbing&amp;nbsp; (if necessary). Many other equipment configurations can be used for tree climbing, depending on the situation and on single operator preferences. We used Pezl equipment but many other companies offer similar products (e.g. Edelrid, Notch, Climbing technologies, DMM, ...). For a complete overview of equipment and companies we recommend a search in google&amp;nbsp; &quot;tree climbing gear&quot; as search keyword. PLEASE NOTE: tree climbing activities should be done only by professionals and are submitted to specific regulatory prescriptions according to the country.</td></tr><tr><td>Avao Sit&lt;br/&gt; (Harness for work positioning and suspension)</td><td>Petzl</td><td>C69AFA 2</td><td></td></tr><tr><td>Rig&lt;br/&gt; (Compact self-braking descender)</td><td>Petzl</td><td>D21A</td><td></td></tr><tr><td>Ascension&lt;br/&gt; (Handled rope clamp for rope ascents)</td><td>Petzl</td><td>B17ALA</td><td></td></tr><tr><td>Eclipse&lt;br/&gt; (Storage for throw-line)</td><td>Petzl</td><td>S03Y</td><td></td></tr><tr><td>Airline&lt;br/&gt; (Throw-line)</td><td>Petzl</td><td>R02Y 060</td><td></td></tr><tr><td>Jet&lt;br/&gt; (Throw-bag)</td><td>Petzl</td><td>S02Y 300</td><td></td></tr><tr><td>Vertex best&lt;br/&gt; (Comfortable helmet for work at height and rescue)</td><td>Petzl</td><td>A10BYA</td><td></td></tr><tr><td>Zillon&lt;br/&gt; (Adjustable work positioning lanyard for tree care)</td><td>Petzl</td><td>L22A 040</td><td></td></tr><tr><td>Ok&lt;br/&gt; (Lightweight oval carabiner)</td><td>Petzl</td><td>M33A SL</td><td></td></tr></tbody></table>

evaluating dryocosmus kuriphilus-induced damage on castanea sativa

Dryocosmus kuriphilus Yasumatsu has become a major pest for Castaneasativa since its arrival in Europe. Its galling activity results in the formation of different gall types and prevents the development of normal shoots. Repeated and uncontrolled attacks cause, besides the production of galls and the attendant gall-related reduction in leaf area, progressive corruption of the branch architecture, including the death of branch parts, and an increase in dormant bud activation. Thus far, there have been few attempts to quantify branch architecture damage. Further, the different methods for assessing infestation degree (MAID) that have been developed focus only on the galls' presence and abundance.
Using the leaf area to sapwood area relationship as a green biomass indicator, we developed in a previous study a damage composite index (DCI) that takes into account the most important branch architectural features, allowing for realistic damage assessment during the entire epidemic process. 
The aim of this study is to present this novel method and highlight differences in the damage description with respect to other broadly used indices. Results show how the DCI depicts branch damage better, especially during the epidemic peak, compared to MAID, which tend to underestimate it. We conclude by suggesting how to properly evaluate the overall impact of the pest by means of our composite damage index, the infestation degree using classic methods, and crown transparency evaluations.

A total of 25 localities in Ticino, Switzerland were visited between 2013 and 2016 in order to create a temporal gradient covering all gall wasp epidemic stages. In total, we collected and analyzed 94 branches in 5 sites at an early epidemic stage (arrival of the pest and beginning of tree damage), 200 branches in 5 sites at the epidemic peak (medium to severe damage due to high level of D. kuriphilus attack), 200 branches in 5 sites at the recovery stage (biocontrol by T. sinensis reached and start of progressive tree recovery), and 54 branches in 5 sites where the pest returned at a very low level over the past 4-5 years. The DCI and two MAID (&#34;average number of galls per bud&#34; (GB)12 and the &#34;proportion of attacked buds&#34; (AB)11) were calculated for each branch. Since the three indices are on different scales, a standardized severity scale from 1 to 9 (1 = very low damage, 9 = extreme damage) is used in order to compare them. DCI is rescaled according to Gehring et al. 20185, GB according to Sartor et al.&#160;201512 and AB according to Gyoutoku and Uemura 198517(Table 2). Comparisons of DCI and MAID within branches were made using the nonparametric Wilcoxon signed-rank test with the Holm adjustment for p values.
At early stages of the epidemic, DCI and MAID are low (median values at 25th and 75th percentiles, DCI = 1 [1, 1], AB = 1 [1, 2], GB = 1 [1, 2]) and express the same level of damage (p &#62; 0.5; Figure 4). During the epidemic peak, the DCI indicates very high damage levels (DCI = 8 [7, 9]) compared to both MAID that indicate only intermediate branch damage level (AB = 4 [3, 5], GB = 4 [3, 6]; Figure 4). Considering branches with low GB and AB values (&#60; 3), 34 out of 71 and 29 out of 59 have more than 30% dead shoots, respectively, whereas from branches with medium GB and AB values (4 and 5), 19 out of 76 and 30 out of 108 have more than 40% dead shoots, respectively. This objectively indicates a high degree of damage. During the recovery phase, differences between DCI and MAID are smaller but still significant (p &#60; 0.01), whereas no differences persist when the recovered phase is reached. Figure 5 provides a visual example of the possible causes of these dissimilarities during the different epidemic stages.
Generally speaking, during the epidemic peak, GB and AB expressed the same damage level as DCI in only 5% of the cases, while tending to underestimate it in ca. 85% of the cases. During the recovery phase, correspondence between GB and DCI and between AB and DCI occurred in 12% and 14% of the cases, respectively. Both MAID underestimated damage in 70% of the cases.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57564/57564fig1.jpg" /> 
Figure 1: Branch damage evaluation illustrating the limitations of classic methods for assessing gallwasp infestation degree (MAID). The sketches show two branches with the same degree of GB (11 galls / 8 buds) and AB (4 attacked buds / 8 buds) but with very different leaf area losses. *Sprout: grown during the current vegetative season; **Shoot: sprout from the previous vegetative season. <a href="/files/ftp_upload/57564/57564fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57564/57564fig2.jpg" /> 
Figure 2: Main features considered when calculating the damage composite index. The dead shoot in the photo is technically a gall on shoot that grew in the previous vegetative season (2016) and completely prevented the elongation of the 2016 shoot. Because the entire gall is dead and no living buds are present on it, it is considered a dead shoot in 2017. <a href="/files/ftp_upload/57564/57564fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57564/57564fig3.jpg" /> 
Figure 3: Essential branch features for assessing the damage composite index (DCI). The dead shoots in (A) and (B) are technically galls on shoots that grew during the previous vegetative season, completely prevented the elongation of the shoot, and are now dead. The dead shoot in (C) died for other causes. The rotten shoot at the bottom of (A) was already dead the year before the dead shoot present on that branch, and is consequently not considered in the DCI calculation. (D), (E) and (F) show different examples of galls on shoots, whereas (G), (H) and (I) represent reactivated dormant buds. <a href="/files/ftp_upload/57564/57564fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57564/57564fig4.jpg" /> 
Figure 4: Evolution of the damage composite index (DCI) and two classic methods for assessing gallwasp infestation degree (MAIDs) across the different epidemic stages. Please refer to Table 2 for more details on the damage categories. DCI: damage composite index (see paragraph 5 of the protocol); AB: proportion of attacked buds (no. attacked buds / no. buds); GB: average number of galls per bud (no. galls / no. buds). Labels on top (n) indicate the number of sampled branches per epidemic stage. Different letters indicate significant differences (p &#60; 0.01) between DCI and MAID branch values at epidemic stages based on the nonparametric Wilcoxon signed-rank test with the Holm adjustment for p values. Outliers are defined as any observation falling outside 1.5 times the interquartile range above or below the upper or lower quartile respectively. Please note that noise has been added to outlier data to avoid excessive overlapping. <a href="/files/ftp_upload/57564/57564fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57564/57564fig5v2.jpg" /> 
Figure 5: Schematic depictions of chestnut branches at different Dryocosmus kuriphilus epidemic stages. At early epidemic stages (A, B), branch architecture is still intact and both the damage composite index (DCI) and MAID (GB and AB) have very low values. Especially during the epidemic peak and recovery stages, branch architecture may become heterogeneously corrupted with different types and degrees of damage. The damage expressed by DCI, GB, and AB can thus be similar (C) or completely different (D) depending on the severity of branch corruption. Finally, at the recovered stage (E, F), DCI and MAID again have similarly low values with the DCI being somewhat more sensitive to previous year damage (dead branches). <a href="/files/ftp_upload/57564/57564fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Epidemic stage</td>
			<td colspan="2">Years since arrival</td>
			<td>Ts%</td>
			<td>Tree</td>
			<td>Branch</td>
		</tr>
		<tr>
			<td></td>
			<td>Dk</td>
			<td>Ts</td>
			<td>%</td>
			<td>n. per site</td>
			<td>n. per tree</td>
		</tr>
		<tr>
			<td>Early</td>
			<td>1-2</td>
			<td>0-1</td>
			<td>0-5</td>
			<td>10</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Peak</td>
			<td>3</td>
			<td>2-3</td>
			<td>&#62;5 &#60;70</td>
			<td>10</td>
			<td>1-2</td>
		</tr>
		<tr>
			<td>Prolonged peak</td>
			<td>4-5</td>
			<td>2-3</td>
			<td>&#62;5 &#60;30</td>
			<td>10</td>
			<td>2-3</td>
		</tr>
		<tr>
			<td>Early recovery</td>
			<td>6-7</td>
			<td>4-5</td>
			<td>&#62;70</td>
			<td>10</td>
			<td>2-3</td>
		</tr>
		<tr>
			<td>Recovery</td>
			<td>8-10</td>
			<td>6-8</td>
			<td>&#62;70</td>
			<td>10</td>
			<td>2-3</td>
		</tr>
		<tr>
			<td>Fully recovered</td>
			<td>&#62;10</td>
			<td>&#62;9</td>
			<td>&#62;70</td>
			<td>10</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
Table 1: Minimum number of trees and branches required based on epidemic stage and Torymus sinensis parasitization rate. Dk = Dryocosmsus kuriphilus; Ts = Torymus sinensis; Ts% = Torymus sinensis parasitization rate calculated as follows: number of living T. sinensis / total number of chambers * 100 (at gall level).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Conversion scale</td>
			<td>DCI</td>
			<td>GB</td>
			<td>AB</td>
		</tr>
		<tr>
			<td rowspan="2">Damage severity</td>
			<td rowspan="2">Points</td>
			<td rowspan="2"></td>
			<td>Average&#160;</td>
			<td rowspan="2">Proportion</td>
		</tr>
		<tr>
			<td>per shoot</td>
		</tr>
		<tr>
			<td>No damage</td>
			<td>1</td>
			<td>&#8804;2.5</td>
			<td>&#8804;0.1</td>
			<td>&#8804;0.1</td>
		</tr>
		<tr>
			<td>Very low</td>
			<td>2</td>
			<td>&#62;2.5 - &#8804;5</td>
			<td>&#62;0.1 - &#8804;0.2</td>
			<td>&#62;0.1 - &#8804;0.2</td>
		</tr>
		<tr>
			<td>Low</td>
			<td>3</td>
			<td>&#62;5 - &#8804;7.5</td>
			<td>&#62;0.2 - &#8804;0.3</td>
			<td>&#62;0.2 - &#8804;0.3</td>
		</tr>
		<tr>
			<td>Mild</td>
			<td>4</td>
			<td>&#62;7.5 - &#8804;10</td>
			<td>&#62;0.3 - &#8804;0.4</td>
			<td>&#62;0.3 - &#8804;0.4</td>
		</tr>
		<tr>
			<td>Moderate&#160;</td>
			<td>5</td>
			<td>&#62;10 - &#8804;15</td>
			<td>&#62;0.4 - &#8804;0.5</td>
			<td>&#62;0.4 - &#8804;0.5</td>
		</tr>
		<tr>
			<td>High</td>
			<td>6</td>
			<td>&#62;15 - &#8804;20</td>
			<td>&#62;0.5 - &#8804;0.6</td>
			<td>&#62;0.5 - &#8804;0.6</td>
		</tr>
		<tr>
			<td>Very High</td>
			<td>7</td>
			<td>&#62;20 - &#8804;25</td>
			<td>&#62;0.6 - &#8804;0.7</td>
			<td>&#62;0.6 - &#8804;0.7</td>
		</tr>
		<tr>
			<td rowspan="2">Extreme</td>
			<td>8</td>
			<td>&#62;25 - &#8804;30</td>
			<td>&#62;0.7 - &#8804;0.8</td>
			<td>&#62;0.7 - &#8804;0.8</td>
		</tr>
		<tr>
			<td>9</td>
			<td>&#62;30</td>
			<td>&#62;0.8</td>
			<td>&#62;0.8</td>
		</tr>
	</tbody>
</table>
Table 2: Conversion scale for the three indices: damage composite index (DCI), number of galls per bud (GB) and attacked buds (AB). DCI is rescaled according to Gehring et al.&#160;20185, GB according to Sartor et al.&#160;201512, and AB according to Gyoutoku and Uemura 198517.
Supplemental File 1. <a href="/files/ftp_upload/57564/Supplemental_File_1.pdf" target="_blank">Please click here to download this file.</a>
Supplemental File 2. <a href="/files/ftp_upload/57564/Supplemental_File_2.pdf" target="_blank">Please click here to download this file.</a>
Supplemental File 3. <a href="/files/ftp_upload/57564/Supplemental_File_3.pdf" target="_blank">Please click here to download this file.</a>
Supplemental File 4. <a href="/files/ftp_upload/57564/Supplemental_File_4.pdf" target="_blank">Please click here to download this file.</a>
Supplemental Coding File 1. <a href="/files/ftp_upload/57564/supplemental_coding_file_1.R" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Evaluating Dryocosmus Kuriphilus-induced Damage on Castanea Sativa at JoVE.com

Evaluating Dryocosmus Kuriphilus-induced Damage on Castanea Sativa

It is common practice to assess the damage caused by Dryocosmus kuriphilus by considering the abundance of galls alone rather than by also taking related branch corruption into consideration. We propose a composite damage index that takes into account the most important branch features, thus enabling more realistic damage assessment.

Evaluating Dryocosmus Kuriphilus-induced Damage on Castanea Sativa

Dryocosmus kuriphilus Yasumatsu has become a major pest for Castaneasativa since its arrival in Europe. Its galling activity results in the formation of ...

evaluating-dryocosmus-kuriphilus-induced-damage-on-castanea-sativa

Nanyang Technological University

Research

JoVE Journal

Environment

7.2K Views.  WSL Swiss Federal Research Institute. Het is gebruikelijk om te beoordelen van de schade veroorzaakt door Dryocosmus kuriphilus door de overvloed aan kurkachtig alleen rekening te houden in plaats van door ook verwante tak corruptie in aanmerking te nemen. Wij stellen een samengestelde schade-index, waarbij rekening wordt gehouden met de belangrijkste kenmerken van de tak, waardoor meer realistische raming van de schade.

Video: Evaluating Dryocosmus Kuriphilus-induced Damage on Castanea Sativa

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The disease is initiated by feeding activities of the invasive beech scale insect, Cryptococcus fagisuga, which creates entry points for infection by one of the Neonectria species of fungus.&#160;Without scale infestation, there is little opportunity for fungal infection. Using scale eggs to artificially infest healthy trees in heavily BBD impacted stands demonstrated that these trees were resistant to the scale insect portion of the disease complex1.&#160;Here we present a protocol that we have developed, based on the artificial infestation technique by Houston2, which can be used to screen for scale-resistant trees in the field and in smaller potted seedlings and grafts.&#160;The identification of scale-resistant trees is an important component of management of BBD through tree improvement programs and silvicultural manipulation.

A Technique to Screen American Beech for Resistance to the Beech Scale Insect Cryptococcus fagisuga Lind.

Beech bark disease is initiated by feeding activities of the beech scale insect that create fungal entry points in the bark.&#160;Trees that are resistant to the scale insect are also disease resistant.&#160;Here we present the protocol we have developed to screen individual beech trees for beech scale resistance.

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The ...

Remote Sensing Evaluation of Two-spotted Spider Mite Damage on Greenhouse Cotton

The objective of this study was to evaluate a ground-based multispectral optical sensor as a remote sensing tool to assess foliar damage caused by the two-spotted spider mite (TSSM), Tetranychus urticae Koch, on greenhouse grown cotton. TSSM is a polyphagous pest which occurs on a variety of field and horticultural crops. It often becomes an early season pest of cotton in damaging proportions as opposed to being a late season innocuous pest in the mid-southern United States. Evaluation of acaricides is important for maintaining the efficacy of and preventing resistance to the currently available arsenal of chemicals and newly developed control agents. Enumeration of spider mites for efficacy evaluations is laborious and time consuming. Therefore, subjective visual damage rating is commonly used to assess density of spider mites. The NDVI (Normalized Difference Vegetation Index) is the most widely used statistic to describe the spectral reflectance characteristics of vegetation canopy to assess plant stress and health consequent to spider mite infestations. Results demonstrated that a multispectral optical sensor is an effective tool in distinguishing varying levels of infestation caused by T. urticae on early season cotton. This remote sensing technique may be used in lieu of a visual rating to evaluate insecticide treatments.

This manuscript describes a multispectral optical sensor that effectively detected damage to early season cotton artificially infested with varying densities of the two-spotted spider mite populations.

The objective of this study was to evaluate a ground-based multispectral optical sensor as a remote sensing tool to assess foliar damage caused by the ...

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide regulation studies. The risk of exposure to fungicide residues is likely to be especially high if the spray occurs on, or near host plants while the bees are collecting pollen to provision their nests. For species of Osmia that consume pollen from a select group of plants (oligolecty), the inability to use pollen from non-host plants can increase their risk factor for fungicide-related toxicity. This manuscript describes protocols used to successfully rear oligolectic mason bees, Osmia ribifloris sensu lato, from egg to prepupal stage within cell culture plates under standardized laboratory conditions. The in vitro-reared bees are subsequently used to investigate the effects of fungicide exposure and pollen source on bee fitness. Based on a 2 &#215; 2 fully crossed factorial design, the experiment examines the main and interactive effects of fungicide exposure and pollen source on larval fitness, quantified by prepupal biomass, larval developmental time, and survivorship. A major advantage of this technique is that using in vitro-reared bees reduces natural background variability and allows the simultaneous manipulation of multiple experimental parameters. The described protocol presents a versatile tool for hypotheses testing involving the suite of factors affecting bee health. For conservation efforts to be met with significant, lasting success, such insights into the complex interplay of physiological and environmental factors driving bee declines will prove to be critical.

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Fungicide sprays on flowering plants may expose solitary bees to high concentrations of pollen-borne fungicide residues. Using laboratory-based experiments involving in vitro-reared bee larvae, this study investigates the interactive effects of consuming fungicide-treated pollen derived from host and non-host plants.

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide ...

Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that improve not only the interpretation of the effects of environmental stressors on organisms but also the determination of their possible impact. It is well known that the use of ecotoxicological biomarkers at different levels of organization allows for the prediction of the biological responses of organisms to environmental stressors, which is useful in environmental risk assessment. 
Nevertheless, it is necessary to consider the optimization of basic procedures, to generate historical data in control groups, and to employ specific bioassays to evaluate responses in organs and tissues in order to elucidate the nature and variation of the effects observed. Therefore, the present work aims to describe several ecotoxicological methodologies employed in all stages of neotropical anurans at different ecological levels and to validate them as useful biomarkers to be used both in wildlife and in laboratory conditions. In this work, these biomarkers were applied at the individual/organismic level (body condition index), histological/physiological level (histopathology, histometric, and pigmentary analyses), biochemical level (oxidative stress enzymes), and genetic level (direct and oxidative damage in DNA by comet assay). 
Although these methodologies have small variations or modifications depending on the species, these techniques provide effective biomarkers for evaluating the effect of xenobiotics on anurans, which possess certain characteristics that make them useful indicator species of aquatic and terrestrial ecosystems. In conclusion, the battery of biomarkers employed in the present study has proven to be adequate for estimating toxic responses in Neotropical anurans and can be further recommended as bioindicators for identifying the impact of pollutants on the aquatic ecosystems of the region. Finally, it is recommended to achieve the standardization of these important biomarkers for anurans in specific regions as well as to possibly include them in risk assessments and decision-making.

In this paper, standardized ecotoxicological methods for the evaluation of biomarkers in neotropical anuran species are presented. Specifically, this paper details several methodologies at different scales of ecotoxicological evaluation, such as the genetic, cellular-histological, biochemical, morphological, and individual levels.

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that ...

Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis

Radiation-induced skin fibrosis (RISF) can result from a plethora of scenarios including cancer therapy, accidental exposure, or acts of terrorism. Radioactive beams can penetrate through the skin and affect the structures in their path including skin, muscles, and internal organs. Skin is the first structure to get exposed to radiation and is susceptible to develop chronic fibrosis, which is challenging to treat. Currently, limited treatment options show moderate efficacy in mitigating radiation-related skin fibrosis. A key factor hindering the development of effective countermeasures is the absence of a convenient and robust model that could allow for translation of the experimental findings to humans. Here, a robust and reproducible murine hind limb skin fibrosis model has been established for prophylactic and therapeutic evaluation of possible agents for functional and molecular recovery.
The right hind limb was irradiated using a single dose of 40 (Gray) Gy to induce skin fibrosis. Subjects developed edema and dermatitis in the early stages proceeded by visible skin constriction. Irradiated limbs showed a significantly reduced limb range of motion in the following weeks. In late stages, acute side effects subsided, yet chronic fibrosis persisted. A gait index was performed as an additional functional assay, which demonstrated the development of functional impairment. These non-invasive methods demonstrated reliable measurements for tracing fibrosis progression, which is supported by histological analyses. The radiation dose, application, and post-irradiation analyses employed in this model offer a vigorous and reproducible method for studying radiation-induced skin fibrosis and testing the efficacy of therapeutical agents.

Here we present a protocol to induce radiation-induced skin fibrosis in the hind limb of mice and perform post-irradiation measurements of chronic impairment via limb excursion and gait index analyses to evaluate the functional outcome. The model elucidates radiation-related skin fibrosis mechanisms and is useful in subclinical therapeutic studies.

Radiation-induced skin fibrosis (RISF) can result from a plethora of scenarios including cancer therapy, accidental exposure, or acts of terrorism. ...

Cancer Research

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

Neuroscience

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

Behavior

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.

Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

We quantify epidermal cell death in frogs with chytridiomycosis using two methods. First, we use terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ histology to determine differences between clinically infected and uninfected animals. Second, we conduct a time series analysis of apoptosis over infection using a caspase 3/7 protein analysis.

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is ...

Immunology and Infection

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Biology

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. ...

Evaluating Dryocosmus Kuriphilus-induced Damage on Castanea Sativa

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video