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 <e...

<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>

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, cl...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 
			(Foldable and lightweight chair)</td>
			<td>Any brand</td>
			<td></td>
			<td>Companies: Kelty, Campz, Half-Ton.</td>
		</tr>
		<tr>
			<td>Felco 9 secateurs 
			(One-hand pruning shear)</td>
			<td>Felco</td>
			<td></td>
			<td>Other companies: Bahco.</td>
		</tr>
		<tr>
			<td>AP-5M-Aluminium Pole 
			(Telescopic tree pruner pole)</td>
			<td>Bahco</td>
			<td>8152079</td>
			<td rowspan="2">Other companies: Spear &#38; Jackson, Kingfisher, Hortex, Fiskars.</td>
		</tr>
		<tr>
			<td>P34-37 top pruner 
			(Telescopic tree pruner head)</td>
			<td>Bahco</td>
			<td>8002787</td>
		</tr>
		<tr>
			<td>100 ft Fiberglass Long Tape 
			(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 
			(Low stretch kernmantel rope, flexible and lightweight for rope access)</td>
			<td>Petzl</td>
			<td>R077AA03</td>
			<td rowspan="10">Basic equipment for tree climbing&#160; (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&#160; &#34;tree climbing gear&#34; 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 
			(Harness for work positioning and suspension)</td>
			<td>Petzl</td>
			<td>C69AFA 2</td>
		</tr>
		<tr>
			<td>Rig 
			(Compact self-braking descender)</td>
			<td>Petzl</td>
			<td>D21A</td>
		</tr>
		<tr>
			<td>Ascension 
			(Handled rope clamp for rope ascents)</td>
			<td>Petzl</td>
			<td>B17ALA</td>
		</tr>
		<tr>
			<td>Eclipse 
			(Storage for throw-line)</td>
			<td>Petzl</td>
			<td>S03Y</td>
		</tr>
		<tr>
			<td>Airline 
			(Throw-line)</td>
			<td>Petzl</td>
			<td>R02Y 060</td>
		</tr>
		<tr>
			<td>Jet 
			(Throw-bag)</td>
			<td>Petzl</td>
			<td>S02Y 300</td>
		</tr>
		<tr>
			<td>Vertex best 
			(Comfortable helmet for work at height and rescue)</td>
			<td>Petzl</td>
			<td>A10BYA</td>
		</tr>
		<tr>
			<td>Zillon 
			(Adjustable work positioning lanyard for tree care)</td>
			<td>Petzl</td>
			<td>L22A 040</td>
		</tr>
		<tr>
			<td>Ok 
			(Lightweight oval carabiner)</td>
			<td>Petzl</td>
			<td>M33A SL</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. si...

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 ...

This method was developed by relating leaf area loss due to the gall wasp attack to the main branch feature such as dormant buds, galls, and dead shoots involved. Considering and combining these feature in a composite damage index allow a realistic evaluation of the tree state during the entire epidemic process, including the recovery phase. The main advantage is that it reveals the real branch alteration even during the recovery phase when the presence of galls becomes poor.We first had the idea for this method when we realized that Dryocosmus kuriphilus attack also causes corruption of the branch architecture beside gall-related reduction in leaf area. Though this method is species specific, the methodology used to create it can be adapted and implemented to any other tree species and related pest. In order to collect the data, arrange for a clipboard, a camping chair, secateurs, a telescopic tree pruner, and a 30-meter measuring tape.Get tree climbing equipment for analysis if the top crown is above eight meters. While collecting, gather three types of branches which represent damaged, healthiest, and an intermediate one to constitute the entire branch diversity. Then, attach the start of the measuring tape near the blade of the telescopic tree pruner.Then use the telescopic pruner to cut the branch. Once the branch is cut, record its cutting height, aspect, and whether the branch type is architectural or reiteration. Next, to gather general information about the branch, assign a unique ID and record the age and the maximum length of the branch from the first branching point to the apex.First look for a sprout, which is a freshly formed structure that has grown during the current vegetative season from a developed bud on a shoot. Next gather an alive shoot, which is a sprout that has developed from the previous vegetative season with respect to the sampling date, then a dead shoot which has died either due to D.kuriphilus attack or naturally. Then collect the reactivated dormant buds which are freshly formed sprouts that have grown during the present vegetative season from a dormant bud on a multi-layer branch part that is older than the shoot.Finally look for a gall on the shoot which usually develops on the base or along the axis of a sprout. Then count and record all the live and dead shoots, reactivated dormant buds, and all the galls on the shoots respectively. To calculate the damage composite index, select the section corresponding to the dead shoot, then calculate the proportion of the dead shoot.Next calculate the proportion of the reactivated dormant bud. After measuring the reactivated dormant bud population, calculate the number of galls on the shoots. Then measure the damage composite index considering the population of dead shoot, reactivated dormant bud, and the number of galls.Next measure the damage severity on the basis of the damage composite index scale. This plot represents early stages of infestation comparing between the DCI and the two MAIDs within the branches. In the plot the Y-axis represents a standardized severity scale where both DCI and the two MAIDs are very low, thereby indicating minimum damage in the early stages.This plot compares the DCI and the two MAIDs during the peak stages of the infestation. The DCI represents a very high damage level when compared to both the MAIDs showing intermediate damage. Interestingly, in the peak and the recovery stages, a heterogeneous branch pattern is observed based on the damage incurred on the basis of the corruption level of the branch.In the case of high corruption level, MAIDs tend to underestimate the real damage because they are only based on the presence of galls. The next two plots represent the DCI and the MAIDs in the recovery and the recovered stages of the infestation. During the recovery stage, the plot shows smaller yet significant differences between the DCI and the two MAIDs.However there isn't any difference between the indices in the recovered stage, with the DCI and the two MAIDs showing somewhat similar values. The damage composite index allows to evaluate the real damage caused by Dryocosmus kuriphilus on the branch architecture. Combining this procedure with other methods, like the Sanasilva crown transparency approach and the traditional method for assessing the infestation degree, ensure a thorough pest assessment.Specifically use this procedure to evaluate the branch architecture damage. Use the traditional method for assessing the infestation degrees such as the proportion of attacked bark to evaluate the pest population and use the Sanasilva crown transparency index for a general crown assessment. After watching this video, you should have a good understanding of how to apply the damage composite index to properly evaluate the impact of Dryocosmus kuriphilus on the branch architecture of the chestnut tree.

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7.1K ビュー.  WSL Swiss Federal Research Institute. This method was developed by relating leaf area loss due to the gall wasp attack to the main branch feature such as dormant buds, galls, and dead shoots involved. Considering and combining these feature in a composite damage index allow a realistic evaluation of the tree state during the entire epidemic process, including the recovery phase. The main advantage is that it reveals the real branch alteration even during the recovery phase when the presence of galls becomes poor.We first h...

ビデオ: Evaluating Dryocosmus Kuriphilus-induced Damage on Castanea Sativa

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce (Lactuca sativa) Germplasm Collections

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely drought-tolerance traits. Fresh water availability for agricultural use is a growing concern across the United States as well as many regions of the world. Short-term drought events along with regulatory intervention in the regulation of water availability coupled with the looming threat of long-term climate shifts that may lead to reduced precipitation in many important agricultural regions has increased the need to hasten the development of crops adapted for improved water use efficiency in order to maintain or expand production in the coming years. This protocol is not meant as a step-by-step guide to identifying at either the physiological or molecular level drought-tolerance traits in lettuce, but rather is a method developed and refined through the screening of thousands of different lettuce varieties. The nature of this screen is based in part on the streamlined measurements focusing on only three water-stress indicators: leaf relative water content, wilt, and differential plant growth following drought-stress. The purpose of rapidly screening a large germplasm collection is to narrow the candidate pool to a point in which more intensive physiological, molecular, and genetic methods can be applied to identify specific drought-tolerant traits in either the lab or field. Candidates can also be directly incorporated into breeding programs as a source of drought-tolerance traits.

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce Lactuca sativa Germplasm Collections

This protocol was developed to screen a large germplasm collection of the leafy vegetable lettuce (Lactuca sativa L.) for drought-tolerance, in order to identify a small candidate pool of lettuce for use in physiological, molecular, and genetic studies to identify underlying drought-tolerance traits along with breeding programs.

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely ...

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ecological interactions. Arthropods are usually tracked in nature by tagging them with a unique mark and then re-collecting them over time and space to determine their dispersal capabilities. In addition to actual physical tags, such as colored dust or paint, various types of proteins have proven very effective for marking arthropods for ecological research. Proteins can be administered internally and/or externally. The proteins can then be detected on recaptured arthropods with a protein-specific enzyme-linked immunosorbent assay (ELISA). Here we describe protocols for externally and internally tagging arthropods with protein. Two simple experimental examples are demonstrated: (1) an internal protein mark introduced to an insect by providing a protein-enriched diet and (2) an external protein mark topically applied to an insect using a medical nebulizer. We then relate a step-by-step guide of the sandwich and indirect ELISA methods used to detect protein marks on the insects. In this demonstration, various aspects of the acquisition and detection of protein markers on arthropods for mark-release-recapture, mark-capture, and self-mark-capture types of research are discussed, along with the various ways that the immunomarking procedure has been adapted to suit a wide variety of research objectives.

Proteins are often used to mark arthropods for dispersal research. Methods for administering internal and external protein marks on arthropods are demonstrated. Protein mark detection techniques, either through indirect or sandwich enzyme-linked immunosorbent assays (ELISAs), are also illustrated.

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ...

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 ...

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a series of incubators. A temperature gradient is passively established across the surface of the table between the heated and cooled ends and is lost quickly at distances above the surface. Since temperature is only controlled on the table surface, experiments are restricted to shallow containers, such as Petri dishes, placed on the table. Welding continuous aluminum vertical strips or gussets perpendicular to the surface of a table enables temperature control in depth via convective heat flow. Soil in the channels between gussets was maintained across a gradient of temperatures allowing a greater diversity of experimentation. The gusseted design was evaluated by germinating oat, lettuce, tomato, and melon seeds. Soil temperatures were monitored using individual, battery-powered dataloggers positioned across the table. LED lights installed in the lids or along the sides of the gradient table create a controlled temperature chamber where seedlings can be grown over a range of temperatures. The gusseted design enabled accurate determination of optimum temperatures for fastest germination rate and the highest percentage germination for each species. Germination information from gradient table experiments can help predict seed germination and seedling growth under the adverse soil conditions often encountered during field crop production. Temperature effects on seed germination, seedling growth, and soil ecology can be tested under controlled conditions in a laboratory using a gusseted thermogradient table.

Traditional thermogradient tables create a range of temperatures across the surface. Welding gussets perpendicular to the surface of a thermogradient table will control temperature in depth increasing possible research applications.

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or concentration-response curve, where mortality increases as the dose or concentration of an insecticide is increased. Standard lethal concentration (LC50) and lethal dose (LD50) tests that result in 50% mortality of a test population can be challenging for evaluating toxicity of insecticides against non-adult insect life stages, such as eggs and early instar or nymphal stages. However, this information is essential for understanding insecticide efficacy in all bed bug life stages, which affects control and treatment efforts. This protocol uses a standard dipping bioassay modified for bed bug eggs and a contact insecticidal assay for treating nymphal first instars. These assays produce a concentration-response curve to further quantify LC50 values for insecticide evaluations.

A Method for Evaluating Insecticide Efficacy against Bed Bug,  Cimex lectularius, Eggs and First Instars

Insecticide evaluations are often targeted against adult insects, rather than immature stages. Here, we present a protocol for evaluating insecticides against bed bug eggs with a comparison to the first nymphal bed bug stage. These protocols could be adjusted for other insects to evaluate insecticide efficacy in non-adult life stages.

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or ...

Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be brought back to the beehive after the bees have foraged on flowers that have been sprayed with pesticides. Pesticide contaminated food can be exchanged between workers which then feed larvae and therefore can potentially affect the development of honey bees. Thus, residual pesticides in the environment can become a chronic damaging factor to honey bee populations and gradually lead to colony collapse. In the presented protocol, honey bee feeding methods are described and applied to either an individual honey bee or to a colony. Here, the insect growth regulator (IGR) pyriproxyfen (PPN), which is widely used to control pest insects and is harmful to the development of honey bee larvae and pupae, is used as the pesticide. The presenting procedure can be applied to other potentially harmful chemicals or honeybee pathogens for further studies.

Herein we present a method to feed pesticide contaminated food to both an individual honey bee and a beehive colony. The procedure evaluates the pesticide effect on individual honey bees by in vivo feeding of basic larval diet and also on the natural condition of beehive colony.

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at capturing and quantifying the dynamics and impact of this invasive insect pest as well as the development of its host plant across a heterogeneous landscape. The cornerstone of this comprehensive monitoring system is timely georeferenced data collection on CBB movement, coffee berry infestation, mortality by the fungus Beauveria bassiana, and coffee plant phenology via a mobile electronic data recording application. This electronic data collection system allows field records to be georeferenced through built-in global positioning systems, and is backed by a network of weather stations and records of farm management practices. Comprehensive monitoring of CBB and host plant dynamics is an essential part of an area-wide project in Hawaii to aggregate landscape-level data for research to improve management practices. Coffee agroecosystems in other parts of the world that experience highly variable environmental and socioeconomic factors will also benefit from implementing this protocol, in that it will drive the development of customized integrated pest management (IPM) to manage CBB populations.

Comprehensive monitoring of coffee berry borer and host plant dynamics is essential for aggregating landscape-level data to improve management of this invasive pest. Here, we present a protocol for scientific monitoring of coffee berry borer movement, infestation, mortality, coffee plant phenology, weather, and farm management via a mobile electronic data recording application.

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at ...

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...