The authors would like to thank the U.S. Department of Agriculture Forest Service for funding this project through USFS Agreement 13-CS-11090800-022. We would like to thank J. Suda, W. Holland, and others for laboratory assistance, and R. Richards for field assistance.

1. Building the sampling device prior to going to the field
<ol>
	<li>Using bolt cutters, large wire cutters, or an electric grinding disk, remove the bottom 1/3 of the 30 cm wire tomato cage so that it is approximately 55 cm in length.</li>
	<li>Cut two, 50 cm braces made from aluminum or similarly semi-rigid material to use as attachment rods and braces on each side of the largest end of the tomato cage. At 38 cm from the end, use a table-top vice or large grasping tool such as channel locks to bend the brace to an approximately 30&#730; angle. Attach the longer end of each of the two attachment rods to opposite sides of the tomato cage with zip ties and duct or electrical tape ensuring the tape is wrapped around at least 6 cm of the cage and rod. Be certain to wrap the tape around the cage and rod numerous times to ensure the cage is permanently attached to the rod.</li>
	<li>Attach the other end of each of the two attachment rods on the opposite sides of the end of an extendable pole with zip ties and duct or electrical tape. As before, wrap the tape multiple times to affix it permanently ensuring the tape overlaps the pole and rods by at least 6 cm. Be certain the opening of the cage is in contact with the end of the telescoping pole when the cage is attached.</li>
	<li>Attach the cage directly onto the end of the pole using zip ties and electrical or duct tape. Attach hook-and-loop fastener strips at 3 points to the opening of the cage 90&#730; from the previously attached pole. 
	NOTE: These strips will be use later to keep the bag open.</li>
</ol>
2. Enclosing the branch
<ol>
	<li>Attach 2&#160;pieces of hook-and-loop fastener to the outside opening of the bag so they align with the hook-and-loop fastener attached to the opening of the cage. These will be used to hold the opening of the bag in place while it is brought over a sample branch. Be certain the hook-and-loop fastener is aligned so when the bag is inserted and attached, the opening to the pull strings of the bag run parallel to the telescoping pole.</li>
	<li>Insert a ~49 L kitchen garbage bag in the wire tomato cage. Place one gator clip on each respective side of the bottom of the bag and attach the clips to both the bag and wire cage to hold the bag against the cage. Repeat the same procedure for the top of the bag with one gator clip attached to the pull string and the wire cage opposite of the pole.&#160;</li>
	<li>Attach para cord to&#160; the bag&#8217;s draw string closest to the pole. Cut four pieces of plastic or hard rubber tubing in 4 cm sections and attach with duct or electrical tape at four locations. The first should be placed on the extending part of the pole about 0.5 m from the end of the pole nearest the tomato cage.&#160; The remaining 3 should be placed equidistance along the bottom section of the extending pole starting about 5 cm from the top of the bottom section (i.e., one each along the top, middle and bottom). Thread the end of the para cord that is not attached to the bag through the plastic tubing.</li>
	<li>For each sample tree, use a random number generator to select a sample height that is within the height of the extension pole when extended at maximum length. Use a random number generator to select a sample distance from the tree trunk. Identify a branch that will fit in the bag with minimal disturbance to the foliage and is the height and distance from the trunk based on the numbers generated from the random number generator.</li>
	<li>Raise the sampling pole to a height parallel with the desired branch. Quickly slide the bag over the branch then rapidly pull the para cord strings attached to the draw strings on the bag to seal the bag. Practice this a few times prior to the first attempt to become efficient at incorporating the foliage with minimal disturbance to the leaves.</li>
	<li>Have a second person clip the branch at the location adjacent to the bag&#39;s opening with the extension pole pruner. Carefully bring the sample bag to the ground and rapidly tie the bag&#39;s draw strings closed. Attempt to complete the bagging, cutting, and bag-tying steps as quickly as possible to prevent insects from escaping.</li>
	<li>Store the bagged branch in a freezer until ready to conduct the laboratory arthropod analysis.</li>
</ol>
3. Arthropod analysis
<ol>
	<li>Hold the frozen bag and branch upright and shake the sample branch while in the bag to dislodge arthropods into the bag. Carefully remove the branch and rinse in large collection pan to remove remaining arthropods. Empty remaining material from the bag into the collection pan. Remove any non-arthropod debris.</li>
	<li>Separate arthropods into desired taxonomic groups. Note differences between larvae and adults.</li>
	<li>Quantify arthropods as desired. If biomass is of interest, either measure length of arthropods and use published length mass table to estimate biomass, or place arthropods in small drying pans, dry in drying oven for 24 h at 45 &#176;C, and weigh on an electronic balance.</li>
</ol>
4. Estimating density
<ol>
	<li>To estimate density and control for variation in leaf structure and leaf density between samples within tree species and among tree species either:</li>
	<li>Count and measure the surface area of the leaves from each sample.</li>
	<li>Dry the leaves in a drying oven for 48 h at 45 &#176;C and weigh the leaves on an electronic balance.</li>
	<li>Measure the length of all woody branch within the sample. 
	NOTE: Diel differences occur in arthropod communities, so sampling should be conducted throughout the entire period of inference.</li>
</ol>

The authors have nothing to disclose.

Two necessities of accurately quantifying arthropod communities are relatively high detection probabilities and known or consistent sampling areas. When sampling for arthropods, less than 100% detection probability can be attributed to either individual arthropods avoiding traps or some individuals that were trapped being undetected during processing. Interceptor traps that intercept flying arthropods (Malaise/window traps, sticky traps, etc.) appear to be the most frequently used approach to enumerate arthropod communities in the forest canopy29,30,31. These types of traps can be placed throughout the canopy, are effective at intercepting flying arthropods, and typically preserve arthropods for long periods (weeks or months) for later identification and quantification29,30,31, though they are typically limited in their ability to trap crawling arthropods31. Interceptor traps that attract arthropods using light or pheromones have additional limitations in that they trap only night flyers and their attractiveness varies with taxon, moonlight, background illumination, and cloud cover impact32,33. Additionally, because arthropods captured in interceptor traps are from unknown distances, the area trapped is unknown. As such, although interceptor traps are effective for indexing flying arthropods across an environmental gradient, data produced from interceptor traps cannot be used to estimate arthropod density25.
An additional method frequently used to monitor foliage arthropods is chemical knockdown34,35. Chemical knockdown can be very effective for collecting diverse groups of arthropods providing accurate estimates of taxonomic richness and diversity. However, this method is expensive and time consuming, is non-specific as it samples all arthropods on the tree including those on the bark and branches, may have unintended environmental impacts due to wind drift, and is illegal in some areas36,37,38,39.
Branch bagging has been demonstrated as an effective method to estimate arthropod density from surface tree foliage with an adequately high capture probability to detect variation across various environmental gradients24,40. The wire tomato cages and 49 L garbage bags used in this study allowed researchers to encompass the branch fully with little to no disturbance prior to the closure of the opening of the bag. As such, it is important that researchers are careful to not disturb foliage of the desired branch sample prior to enclosing it with the sampling bag. Thus, a critical step is to bring the sampling bag parallel with the desired sampling bag and swiftly enclose, seal, and tie the bag after each sample is collected. Sample collection is limited to the maximum height the researcher can hold an extended telescoping pole at (8 m in our study), although the same branch-bagging equipment and methodology can be used in other situations such as suspension in the canopy. Some authors have suggested that when using this procedure active, flighted-arthropods are underrepresented40,41,42. However, we believe that as long as the foliage remains undisturbed until it is enclosed by the sampling bag, it is unlikely that a substantial number arthropods present in or on the foliage at the time escaped capture. The results of our study support this assertion in that when sampling a reasonable number of trees (323), the standard error was at most 17% of the relative arthropod biomass mean (Cayra = 11%, Acer = 12%, Fagus = 17%, Liriodendrum = 15%, and Quercus = 11%). Similarly, when considering guild richness and diversity, the most variable estimate was diversity on Fagus, with a standard error that was 7% of the mean. Clearly these estimates provided adequate precision to model differences among tree genera groups as well as other ecological or environmental variables. A limitation to our results, however, is that although we are confident that detection probability with this method is high, i.e., likely nearing 100%, we do not have a method of independently verifying this assertion. Thus, while we demonstrated detection probability is adequate to detect variation across an environmental variable, which in this case was tree genera, biomass estimates produced from this methodology have the potential to be biased low by some unknown amount40.
Most authors have examined the content of the bag in the field36,42,43,44,45. We believe a critical step to maximizing detection is to freeze the bag as we did, then examine and quantify the content in the laboratory under controlled conditions. We believe this approach will decrease measurement error by minimizing the number of trapped arthropods that are overlooked or are misidentified.
Estimating the area sampled for comparison of density among tree species may be problematic if leaf structure varies considerably among tree species, as was the case in our study. In past studies, when authors were interested in quantifying foliage-dwelling arthropods, they often estimated sampling area by weighing leaves to estimate the amount of substrate available for arthropods46,47,48. The various species of oak trees, however, tend to have thicker waxy leaf cuticles than other tree species. Thus, the mass to surface area ratio for oaks is greater than other species49. Because the mass to surface area ratio is greater in oaks, using the mass of leaves as an estimate of substrate for foliage dwelling arthropods would overestimate sampling area and underestimate arthropod density for oak trees relative to tree species with less thick leaf cuticles. Additionally, if the ability to support arthropods varies among tree species, the surface area of the landscape covered by a given tree species will dictate the level of substrate supported within a specified landscape. Because the amount of surface area a given tree occupies is determined by crown spread (i.e., branch spread outwards from the trunk) and leaf density varies among trees, we believe when quantifying arthropods for consumption by insectivores, total branch length sampled is more appropriate than leaf biomass when estimating total area sampled. Our results again appear to support this assertion in that we detected differences among tree groups consistent with the predicted pattern based on previous studies25. We believe arthropod abundance or biomass per measure of branch length is most appropriate when the primary objective is to compare resources provided for insectivores among tree species. If, however, individuals are comparing tree species that produce leaves with similar leaf cuticle thickness, using leaf biomass as an estimate of sampling area may be more appropriate. Regardless of whether researchers use actual leaf area, leaf area as estimated by leaf biomass, or total branch length as a quantifiable metric, by using the bagging technique, a measurable quantity of arthropods at a specific point in time on a measurable surface area is captured per sample. This allows researchers to use leaf surface area, leaf area as estimated by leaf biomass, or total branch length as a quantifiable metric. This method provides a consistent estimate for comparing quantified arthropods among spatial or temporal variables and an estimate of arthropod density25.
In general, the sampling method described in this article appears to be effective at allowing for spatial or temporal comparisons of foliage-dwelling arthropod metrics. This approach is affordable and feasible at the landscape scale. Furthermore, although freezing the entire branch requires substantial freezer space, freezing the branch then rinsing the branch in water is an effective way to separate arthropods from foliage with minimal effort, therefore providing a cost-efficient approach to obtaining arthropod metrics. Finally, because the primary objective of our original study was to better understand how mesophication of southeastern deciduous forests is likely to impact forest-dwelling insectivorous birds and mammals we grouped arthropods into guilds based on diagnostic morphological features. However, we do not see a reason why these capture techniques cannot be used to quantify arthropods at the species or any other taxonomic level.

Terrestrial arthropods play an important role in our ecosystem. In addition to being of scientific interest arthropods can be both detrimental and beneficial to crops, horticultural plants, and natural vegetation as well as provide an important trophic function in food webs. Thus, understanding the factors that influence arthropod community development and abundance is critical to farmers, pest control managers, plant biologists, entomologists, wildlife ecologists, and conservation biologists that study community dynamics and manage insectivorous organisms. Understanding factors that influence arthropod communities and abundances often requires the capture of individuals. Capture techniques can generally be categorized into qualitative techniques that only detect presence of a species for estimates of species range, richness, and diversity, or semi-quantitative and quantitative techniques that allow for an index or estimate of abundance and density of individuals within a taxonomic group.
Qualitative techniques that only allow inference regarding presence of a species or community structure have an unknown or intrinsically low detection probability or are lacking in providing inference regarding detection probability and size of area sampled. Because detection probability with these techniques is low, variability associated with detection precludes adequate precision for inferring how explanatory variables influence arthropod population metrics. Qualitative techniques used to estimate presence include suction sampling1, light traps2, emergence traps3, feeding patterns on roots4, brine pipes5, baits6, pheromone3, pitfall traps7, Malaise traps8, window traps9, suction traps10, beating trays11, spider webs12, leaf mines, frass13, arthropod galls14, vegetation and root damage15.
Alternatively, semi-quantitative and quantitative techniques allow researchers to estimate or at least consistently sample a specified sample area and estimate probability of detection or assume detection probability is non-directional and adequate as to not obscure the researcher&#39;s ability to detect spatial or temporal variation in abundance. Semi-quantitative and quantitative techniques include sweep nets16, suction or vacuum sampling17, systematic counting of visible arthropods18, sticky traps19, various pot-type traps20, entrance or emergent holes21, chemical knockdown22, sticky and water filled color traps23, and branch bagging and clipping24.
Recent anthropogenic-induced changes to climate and disturbance regimes have led to dramatic changes in plant communities, making interactions between plant-community species composition and arthropod communities an active area of study. Understanding how arthropod communities vary with plant species composition is a critical component for understanding the potential economic and environmental impacts of changes to plant communities. Semi-quantitative or quantitative methods of quantifying arthropod abundance with adequate precision to detect differences among species of plants are needed. In this article, we describe a method for indexing foliage-dwelling arthropods that, with reasonable effort, provided adequate precision to identify differences in individual abundance and biomass, diversity, and richness among 5 taxa of trees commonly found in the southeastern deciduous forests of North America25. This approach provided precision adequate for estimating abundance to allow inference as to how changes in species composition of forest plant communities due to anthropic modified disturbance regimes influence composition of arthropods, potentially influencing abundance and distribution of higher trophic insectivorous birds and mammals. More specifically, by using a modified bagging technique first described by Crossley et al.24, we estimated density of surface, foliage-dwelling arthropods and tested the prediction that we would detect differences in diversity, richness, and abundance of arthropods in the foliage of faster growing more xeric species of trees relative to slower growing more mesic species. The goal of this article is to provide detailed instructions of the technique.
We conducted the study on the Shawnee National Forest (SNF) in southern Illinois. The SNF is a 115,738-ha forest located in the Central Hardwoods region of the Ozarks and Shawnee Hills natural divisions26. The forest comprises a mosaic of 37% oak/hickory, 25% mixed-upland hardwoods, 16% beech/maple, and 10% bottomland hardwoods. The SNF is dominated by second growth oak/hickory in upland xeric areas and sugar maple, American beech, and tulip tree (Liriodendron tulipifera) in sheltered mesic valleys27,28.
Site selection for this method will be dependent on the overarching goals of the study. For example, the primarily goal of our original study was to provide insight into how changes in tree community might influence higher trophic organisms by comparing foliage-dwelling arthropod community metrics between mesic and xeric adapted tree communities. Thus, our primary objective was to quantify the arthropod community on individual trees located within the xeric or mesic tree community. We selected 22 study sites along an oak/hickory (xeric) to beech/maple (mesic) dominated gradient using USFS stand cover maps (allveg2008.shp) in ArcGIS 10.1.1. To prevent potential confounding effects, we selected sites using the following criteria: not located in riparian areas, &#8805;12 ha, and located within contiguous upland-deciduous forest habitat (i.e., elevation above 120 m). All sites contained mature trees &#62;50 years old in hilly terrain, thus comprised similar slopes and aspects. While beech/maple site boundaries were distinguished based on the transition of tree communities, oak/hickory site boundaries were identified artificially using SNF cover maps and ArcGIS 10.1.1. All sites were large forest blocks within un-glaciated terrain; their differences in tree species composition were not due to differences in location on the landscape but were representative of past land usage (e.g., clear cuts or selective harvest). We ground-truthed the maps by uploading discrete polygon shapefiles of each study site to a handheld Global Positioning System (GPS) and verifying tree species composition. We randomly selected sampling points (n = 5) at each site. At each point, we sampled three trees from 0600&#8722;1400 hours during 23 May to 25 June 2014. To locate sample trees, we searched outward to a 30 m radius from vegetation points until mature trees (&#62;20 cm d.b.h.) with branches low enough to sample were found. Typically, the three mature trees that represented three of the five genera (Acer, Carya, Fagus, Liriodendron, and Quercus) of interest and were closest to the center point were sampled.

<table><tbody><tr><td>13 gallon garbage bags</td><td>Glad</td><td>78374</td><td></td></tr><tr><td>Aluminum rod</td><td>Grainger</td><td>48ku20</td><td></td></tr><tr><td>Pruner</td><td>Bartlet arborist supply</td><td>pp-125b-2stick</td><td></td></tr><tr><td>Telescoping pole</td><td>BES</td><td>TPF620</td><td></td></tr><tr><td>Tomato Cage</td><td>Gilbert and Bennet</td><td>42 inch galvanized</td><td></td></tr></tbody></table>

a method for quantifying foliage-dwelling arthropods

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimates of density requires a method with high detection probability and a known sampling area. While most described methods provide a qualitative or semi-quantitative estimate adequate for describing species presence, richness, and diversity, few provide an adequately consistent detection probability and known or consistent sampling areas to provide an index or estimate with adequate precision to detect differences in abundance across environmental, spatial, or temporal variables. We describe how to quantify leaf-dwelling arthropods by sealing the leaves and end of branches in a bag, clipping and freezing the bagged material, and rinsing the previously frozen material in water to separate arthropods from the substrate and quantify them. As we demonstrate, this method can be used at a landscape scale to quantify leaf-dwelling arthropods with adequate precision to test for and describe how spatial, temporal, environmental, and ecological variables influence arthropod richness and abundance. This method allowed us to detect differences in density, richness, and diversity of leaf-dwelling arthropods among 5 genera of trees commonly found in southeastern deciduous forests.

We collected 626 samples from 323 individual trees composing 5 tree groups. For estimates of total arthropod biomass per meter of branch sampled, the standard error ranged from 12% to 18% of the mean for the 5 tree groups (Table 1). This level of precision was adequate to detect variation among tree groups and a quadratic change in biomass with date25. This technique provided more precision when estimating guild diversity as demonstrated by the standard error of arthropod guild diversity (H&#39;) ranging from 3% to 7% of the mean diversity across the 5 tree groups (Table 1). Precision at this level was adequate to detect variation across the 5 tree groups25. Precision of estimates of richness was also very good as demonstrated by standard errors that ranged from 3% to 7% of the mean richness among the 5 tree groups (Table 1). This level of precision was adequate to identify variation among tree groups, a quadratic association with date, decrease in richness with height on the tree, and a positive relationship between arthropod richness and distance from the tree trunk25.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Tree species</td>
			<td colspan="3">Richness</td>
			<td colspan="3">Biomass</td>
			<td colspan="3">Shannon Diversity</td>
		</tr>
		<tr>
			<td>X</td>
			<td>SE</td>
			<td>% of mean</td>
			<td>X</td>
			<td>SE</td>
			<td>% of mean</td>
			<td>X</td>
			<td>SE</td>
			<td>% of mean</td>
		</tr>
		<tr>
			<td>Maple spp. (N = 140)</td>
			<td>3.54</td>
			<td>0.17</td>
			<td>5%</td>
			<td>0.003</td>
			<td>0.0004</td>
			<td>13%</td>
			<td>0.86</td>
			<td>0.05</td>
			<td>6%</td>
		</tr>
		<tr>
			<td>Hickory spp. (N = 141)</td>
			<td>4.62</td>
			<td>0.20</td>
			<td>4%</td>
			<td>0.013</td>
			<td>0.002</td>
			<td>15%</td>
			<td>1.10</td>
			<td>0.04</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>Tulip Poplar (N = 70)</td>
			<td>4.32</td>
			<td>0.20</td>
			<td>5%</td>
			<td>0.011</td>
			<td>0.002</td>
			<td>18%</td>
			<td>1.12</td>
			<td>0.05</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>American Beach (N = 67)</td>
			<td>3.23</td>
			<td>0.22</td>
			<td>7%</td>
			<td>0.002</td>
			<td>0.0003</td>
			<td>15%</td>
			<td>0.81</td>
			<td>0.06</td>
			<td>7%</td>
		</tr>
		<tr>
			<td>Oak spp. (N = 208)</td>
			<td>4.77</td>
			<td>0.15</td>
			<td>3%</td>
			<td>0.006</td>
			<td>0.0007</td>
			<td>12%</td>
			<td>1.10</td>
			<td>0.03</td>
			<td>3%</td>
		</tr>
	</tbody>
</table>
Table 1: Parameter estimates from most parsimonious model25. The mean (X), standard error of the mean (SE), and percentage of the mean of the standard error for each community metric of foliage dwelling-arthropods captured on 5 groups of trees using the described branch clipping method in the Shawnee National Forest in southern Illinois.

Watch this Scientific Journal Video about A Method for Quantifying Foliage-Dwelling Arthropods at JoVE.com

A Method for Quantifying Foliage-Dwelling Arthropods

We describe how to quantify leaf dwelling arthropods by sealing the leaves and end of branches in a bag, clipping and freezing the bagged material, and rinsing the previously frozen material in water to separate arthropods from the substrate for quantification.

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimates of density ...

a-method-for-quantifying-foliage-dwelling-arthropods

Nanyang Technological University

Research

JoVE Journal

Environment

5.8K Views.  Southern Illinois University. Het belangrijkste voordeel van dit protocol is het stelt onderzoekers in staat om zowel vliegende als kruipende geleedpotigen vast te leggen van het gebladerte. Hoewel we deze techniek gebruiken op bomen kan het ook worden gebruikt op struiken en struiken. Wees voorzichtig om te oefenen zakken takken snel zonder verstoring van het gebladerte, zodat actieve geleedpotigen niet ontsnappen vangen.Om te beginnen met deze procedure, gebruik bout snijders, grote draadsnijders, of een elektris...

Video: A Method for Quantifying Foliage-Dwelling Arthropods

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens

Many insects live above the ground as larvae and adults and as pupate below the ground. Compared to the above-ground stages of their life cycles, less attention has been paid on how environmental factors affect these insects when they pupate within the soil. The tea looper, Ectropis grisescens Warren (Lepidoptera: Geometridae), is a severe pest of tea plants and has caused huge economic losses in South China. The protocols described here aim to investigate, through multiple-choice bioassays, whether mature last-instar E. grisescens larvae can discriminate soil variables such as the substrate type and moisture content, and determine, through no-choice bioassays, the impact of the substrate type and moisture content on pupation behaviors and the emergence success of E. grisescens. The results would enhance the understanding of the pupation ecology of E. grisescens and may bring insights into soil-management tactics for suppressing E. grisescens populations. In addition, these bioassays can be modified to study the influences of various factors on the pupation behaviors and survivorship of soil-pupating pests.

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens

Here, we present a protocol to investigate the pupation preference of mature larvae of Ectropis grisescens in response to soil factors (e.g., substrate type and moisture content) using choice bioassays. We also present a protocol of no-choice bioassays to determine the factors that affect the pupation behaviors and survivorship of E. grisescens.

Many insects live above the ground as larvae and adults and as pupate below the ground. Compared to the above-ground stages of their life cycles, less ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

Quantifying Corticolous Arthropods Using Sticky Traps

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimate of density requires a method with high detection probability and a consistent sampling area. We used manufactured sticky traps to compare abundance, total length (a surrogate for biomass), richness, and Shannon diversity of corticolous arthropods among the boles of 5 tree species. Efficacy of this method was adequate to detect variation in corticolous arthropods among tree species and provide a standard error of the mean that was &#60;20% of the mean for all estimates with sample sizes from 7 to 15 individual trees of each species. Our results indicate, even with these moderate sample sizes, the level of precision of arthropod community metrics produced with this approach is adequate to address most ecological questions regarding temporal and spatial variation in corticolous arthropods. Results from this method differ from other quantitative approaches such as chemical knockdown, visual inspection, and funnel traps in that they provide an indication of corticolous arthropod activity over a relatively long-term, better including temporary bole residents, flying arthropods that temporarily land on the tree bole and crawling arthropods that use the tree bole as a travel route from the ground to higher forest foliage. Furthermore, we believe that commercially manufactured sticky traps provide more precise estimates and are logistically simpler than the previously described method of directly applying a sticky material to tree bark or applying a sticky material to tape or other type of backing and applying that to the tree bark.

We describe a semi-quantitative approach of measuring characteristics of corticolous (bark-dwelling) arthropod communities. We placed commercially manufactured sticky traps on tree boles to estimate abundance, total length (a surrogate to biomass), richness, and Shannon diversity for comparison among tree species.

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimate of density ...

Low-Cost Automated Flight Intercept Trap for the Temporal Sub-Sampling of Flying Insects Attracted to Artificial Light at Night

Sampling methods are selected depending on the targeted species or the spatial and temporal requirements of the study. However, most methods for passive sampling of flying insects have a poor temporal resolution because it is time-consuming, costly and/or logistically difficult to perform. Effective sampling of flying insects attracted to artificial light at night (ALAN) requires sampling at user-defined time points (nighttime only) across well-replicated sites resulting in major time and labor-intensive survey effort or expensive automated technologies. Described here is a low-cost automated intercept trap that requires no specialist equipment or skills to construct and operate, making it a viable option for studies that require temporal sub-sampling across multiple sites. The trap can be used to address a wide range of other ecological questions that require a greater temporal and spatial scale than is feasible with previous trap technology.

To study the impacts of artificial light at night (ALAN) on nocturnal flying insects, sampling needs to be confined to nighttime. The protocol describes a low-cost automated flight intercept trap that allows researchers to sample at user-defined periods with increased replication.

Sampling methods are selected depending on the targeted species or the spatial and temporal requirements of the study. However, most methods for passive ...

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2. Plant litter comprises the majority of detritus3, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9 because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11.
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira), a dominant grasshopper herbivore (Melanoplus femurrubrum),and a variety of grass and forb plants9.

We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.

The quantity and quality of detritus entering the soil  determines the rate of decomposition by microbial communities as well as  recycle rates of ...

Biology

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Collecting Hemolymph from Small Arthropods

A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods

Arthropods are known to transmit a variety of viruses of medical and agricultural importance through their hemolymph, which is essential for virus transmission. Hemolymph collection is the basic technology for studying virus-vector interactions. Here, we describe a novel and simple method for the quantitative collection of hemolymph from small arthropods using Laodelphax striatellus (the small brown planthopper, SBPH) as a research model, as this arthropod is the main vector of rice stripe virus (RSV). In this protocol, the process begins by gently pinching off one leg of the frozen arthropod with fine-tipped tweezers and pressing the hemolymph out of the wound. Then, a simple micropipette consisting of a capillary and a pipette bulb is used to collect the transudative hemolymph from the wound according to the principle of capillary forces. Finally, the collected hemolymph can be dissolved into a specific buffer for further study. This new method for collecting hemolymph from small arthropods is a useful and efficient tool for further research on arboviruses and vector-virus interactions.

Author Spotlight: A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods  

We describe a method to collect quantifiable hemolymph efficiently from small arthropods for subsequent analysis.

Arthropods are known to transmit a variety of viruses of medical and agricultural importance through their hemolymph, which is essential for virus ...

Quantification of Arbuscular Mycorrhizal Fungi Colonization Rate in the Study of Invasive Alien Plants

Arbuscular mycorrhizal fungi (AMF) are widely distributed soil fungi in ecosystems and can form symbiotic associations (mycorrhizae) with the roots of most terrestrial plants. Plants provide carbon sources to AMF through mycorrhizal associations, while AMF hyphae can expand the range of nutrient absorption by roots and promote plant nutrient uptake. There are many different species of AMF, and the symbiotic relationships between different species of AMF and different plants vary. Invasive plants can enrich AMF species with better symbiotic capabilities through root exudates, promoting their growth and thereby increasing their colonization in invasive plant roots. At the same time, invasive plants can also disrupt the symbiotic relationship between AMF and native plants, affecting the local plant community, which is one of the mechanisms for successful plant invasion. The colonization rate of AMF in the roots of invasive and native plants indirectly reflects the role of AMF in the process of invasive plant invasion. In this method, collected plant roots can be processed directly or saved in a fixative for later batch processing. Through decolorization, acidification, staining, and destaining treatment of roots, the hyphae, spores, and arbuscular structures of AMF in the root system can be clearly observed. This method can be completed in a basic laboratory to observe and calculate the colonization rate of AMF in the root systems of invasive plants.

This protocol provides a simple and easy-to-use approach for determining the colonization rate of Arbuscular mycorrhizal fungi (AMF) in the roots of invasive plants.

Arbuscular mycorrhizal fungi (AMF) are widely distributed soil fungi in ecosystems and can form symbiotic associations (mycorrhizae) with the roots of ...

A Method for Quantifying Foliage-Dwelling Arthropods

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