A Method for Quantifying Foliage-Dwelling Arthropods

Summary

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.

Abstract

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.

Introduction

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'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 valleys^27,28.

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, ≥12 ha, and located within contiguous upland-deciduous forest habitat (i.e., elevation above 120 m). All sites contained mature trees >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 0600−1400 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 (>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.

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Protocol

1. Building the sampling device prior to going to the field

1. 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.
2. 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˚ 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.
3. 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.
4. 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˚ from the previously attached pole.
   NOTE: These strips will be use later to keep the bag open.

2. Enclosing the branch

1. Attach 2 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.
2. 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. 
3. Attach para cord to  the bag’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.  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.
4. 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.
5. 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.
6. Have a second person clip the branch at the location adjacent to the bag's opening with the extension pole pruner. Carefully bring the sample bag to the ground and rapidly tie the bag's draw strings closed. Attempt to complete the bagging, cutting, and bag-tying steps as quickly as possible to prevent insects from escaping.
7. Store the bagged branch in a freezer until ready to conduct the laboratory arthropod analysis.

3. Arthropod analysis

1. 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.
2. Separate arthropods into desired taxonomic groups. Note differences between larvae and adults.
3. 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 °C, and weigh on an electronic balance.

4. Estimating density

1. To estimate density and control for variation in leaf structure and leaf density between samples within tree species and among tree species either:
2. Count and measure the surface area of the leaves from each sample.
3. Dry the leaves in a drying oven for 48 h at 45 °C and weigh the leaves on an electronic balance.
4. 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.

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Results

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 sta...

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Discussion

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 communit...

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Materials

Name	Company	Catalog Number	Comments
13 gallon garbage bags	Glad	78374
Aluminum rod	Grainger	48ku20
Pruner	Bartlet arborist supply	pp-125b-2stick
Telescoping pole	BES	TPF620
Tomato Cage	Gilbert and Bennet	42 inch galvanized