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Method Article

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

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DOI:

10.3791/53693

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January 28th, 2016

 , 
1USDA-ARS, Arid-Land Agricultural Research Center

In This Article

Summary

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.

Abstract

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.

Introduction

Tracking the movement of arthropod pests, natural enemies (parasitoids and predators), and pollinators in nature is essential for better understanding how to improve ecosystem services. The key component for most types of dispersal research is having a reliable method to tag the arthropod(s) of interest. A variety of materials (e.g., paints, dyes, colored dusts, tags, rare elements, proteins) have been used to mark arthropods to assess their population dynamics, dispersal capabilities, feeding behaviors, and other ecological interactions1,2.

The appropriateness of a marker used for any given dispersal research will be dependent on the type of study being conducted. There are three broad categorizations for marking arthropods: (1) mark-release-recapture (MRR), (2) mark-capture, and (3) self-mark-capture. For mark-release-recapture research, the investigator typically marks the arthropods collectively in the laboratory and releases them at a central point in the field. The arthropods are then recaptured at various spatial and temporal intervals using different collection devices (e.g., sweep net, vacuum, sticky trap)3,4,5. The recaptured specimens are then examined for the specific mark to distinguish released from native individuals. For mark-capture research, the investigator usually applies the mark directly in the field using spray equipment (e.g., backpack sprayer, boom and nozzle sprayer). The best markers for mark-capture research are inexpensive and easily applied to the arthropod's habitat. For self-mark-capture research, the investigator usually applies marks to an arthropod bait6,7 or nest entrance8. In turn, the arthropod marks itself internally by devouring the marked bait or externally by "brushing" up against the mark as it exits the nest.

As mentioned above, many types of markers have been used to tag a variety of arthropod species. However, very few are useful for all three of these dispersal research categories. The development of the protein immunomarking procedure was a major breakthrough for marking insects. Immunomarking puts a protein label on arthropods either internally or externally which, in turn, is detected by an anti-protein specific enzyme-linked immunosorbent assay (ELISA). The first such protein markers used were rabbit immunoglobulin (IgG) and chicken IgG/IgY9,10. They proved to be very effective marks for MRR and self-mark-capture research (see discussion). Unfortunately, IgG/IgY proteins are costly and are therefore not practical for mark-capture research and most types of self-mark-capture research. Subsequently, second-generation protein detection ELISAs were developed for proteins contained in chicken egg whites (albumin), cow's milk (casein) and soy milk (trypsin inhibitor protein). Each assay is highly sensitive, specific, and, most importantly, uses proteins that are much less expensive than the IgG/IgY proteins11. These proteins have proven effective for MRR, mark-capture, and self-mark-capture research (see discussion).

In this article, we describe and demonstrate how to conduct protein mark laboratory retention studies. Such studies are the first phase of research needed for any type of field dispersal study. Specifically, it is critical that investigators know how long the mark will be retained on the targeted arthropod species prior to embarking on field dispersal studies. Here, we describe and demonstrate how to internally and externally mark insects for MRR, mark-capture, and self-mark-capture type field studies. We then demonstrate how to detect the presence of the marks with indirect and sandwich ELISAs.

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Protocol

1. Internal Mark, Retention, and Detection Procedure

  1. Internal marking procedure
    1. Collect insects of interest (n ≈ 100 individuals) from a laboratory colony reared on an artificial diet or from the field and divide into two clean rearing containers.
    2. Place a regular 20 ml diet packet (unmarked negative control treatment) into one of the containers. Supplement a second 20 ml artificial diet packet with 1.0 ml of a 1.0 mg/ml chicken IgG/IgY solution, mix thoroughly, and place it in the other container.
      Note: If the insect of interest does not have an artificial diet, the mark can be placed on or in whatever diet they are normally sustained on (e.g., green bean, moth eggs, water, sugar, honey).
    3. Allow the insects to resume their feeding activity for 24 hr.
  2. Internal mark retention procedure
    1. Remove the diet packets from each container and supply the insects with another food source to sustain them over the duration of the experiment (e.g., green beans).
    2. Collect a cohort (n = 20) of insects daily (or at any desired time interval) from each treatment and freeze immediately at -20 °C.
  3. Sandwich ELISA procedure
    1. Add 50 µl of rabbit anti-chicken IgY primary antibody diluted 1:500 in Tris buffered saline (TBS) to each well of a clean ELISA microplate and incubate for a minimum of 1 hr at RT (note: the microplates can also be incubated O/N at 4 °C).
    2. Discard the antibody from the plate and block each well with 300 µl of a 1.0% non-fat dry milk solution for 30 min.
    3. While waiting for the previous incubation steps, place frozen insects individually in 1.6 ml microcentrifuge tubes with 1.0 ml of TBS.
    4. Grind each insect with a clean pestle until thoroughly homogenized and add a 100 µl of each sample to an individual well of the ELISA plate. Allow the samples to incubate at RT for 1 hr.
    5. Add 100 µl of negative (TBS only) and positive (egg whites in TBS) controls to wells in the first column of each ELISA plate. Also, add 100 µl of negative insect controls (unmarked insects) to the 8 wells in the last column of each plate (Figure 1). Allow the control samples to incubate at RT for 1 hr. Note that the template provided in Figure 1 is the template we use for our standardized assays. The placement of the samples in the wells is up to the discretion of the researcher.
    6. Wash each well three times with phosphate buffered saline (PBS)-tween and then add 50 µl of rabbit anti-chicken IgG/IgY conjugated with horseradish peroxidase diluted 1:10,000 in 1% milk solution and incubate for 1 hr at RT.
    7. Wash each well three times with PBS-tween and add 50 µl of tetramethylbenzidine (TMB) substrate to each well.
    8. After 10 min, read the wells with a microplate spectrophotometer set at a wavelength of 650 nm.

Microplate layout for ELISA assay; samples, controls; data analysis, immunoassay result assessment.
Figure 1. A schematic diagram showing the standardized well designations used for a typical 96 well ELISA microplate. Each plate contains 7 wells dedicated to Tris-buffered saline negative controls (TBS), a well dedicated to a positive protein control (Pos Ctrl), 80 individual wells dedicated to recaptured insects (Sample 1…80), and 8 wells dedicated to unmarked (negative) insect controls (Neg Ctrl 1…8). Please click here to view a larger version of this figure.

2. External Mark, Retention, and Detection Procedure

  1. External marking procedure
    1. Collect insects of interest (n ≈ 100 individuals) from a laboratory colony or from the field and place into two 1.0 L plastic containers with a 2.5 cm diameter hole punched out of the side of each container.
    2. Spray the insects in the first container with 1.0 ml of dH2O (negative control treatment) using a medical nebulizer. Spray the insects in the second container with 1.0 ml of a 5.0% solution of chicken egg whites using a medical nebulizer.
    3. Attach the hose of the nebulizer to a standard laboratory air outlet and then insert the mouth of the nebulizer into the 2.5 cm hole of the container. Then, turn on the air outlet gradually until the nebulizer produces a vapor.
    4. Continue spraying (marking) until the egg whites solution has been dispensed (approx. 2-5 min).
    5. Remove the nebulizer and place a cork into the hole of the plastic container. Allow the insects to stand at RT until the solution has dried completely.
  2. External mark retention procedure
    1. Place each treatment of the dry insects into a separate clean rearing container to avoid further exposure to the mark.
    2. Add food and water to the clean containers to sustain the insects over the duration of the study.
    3. Collect a cohort (n = 20) of insects from each treatment daily and freeze immediately at -20 °C.
  3. Indirect ELISA procedure
    1. Place the frozen insects individually into 1.6 ml microcentrifuge tubes and add 1.0 ml of TBS.
    2. Soak the samples for 1 hr on an orbital shaker set at 120 rpm.
    3. Add negative and positive controls as described in Step 1.3.5.
    4. After soaking, pipette a 100 µl aliquot from each sample into one of the 80 designated sample wells on a new 96 well ELISA plate as shown in Figure 1. Allow the samples to incubate for 1 hr at RT (note: the microplates can also be incubated O/N at 4 °C).
    5. Wash the wells three times with PBS-tween and then add 300 µl of phosphate buffered saline containing 1% bovine serum albumin (PBS-BSA) to each well.
    6. After 30 min at RT wash twice with PBS-tween.
    7. Add 50 µl of rabbit anti-egg albumin primary antibody diluted 1:8,000 in PBS-BSA-Silwet (0.05%) to each well and incubate for 1 hr at RT.
    8. Wash each well three times with PBS-tween and then add 50 µl of goat anti-rabbit IgG conjugated with horseradish peroxidase diluted 1:2,000 in PBS-BSA-Silwet (0.05%) for 1 hr at RT.
    9. Wash each well three times with PBS-tween and add 50 µl of TMB substrate to each well.
    10. After 10 min, read the wells with a microplate spectrophotometer set at a wavelength of 650 nm.

3. Data Analysis 

  1. Calculate the mean absorbance value of the 8 negative control insects on each ELISA plate and then calculate the standard deviation based on the pooled negative controls from all of the ELISA plates of a given study12.
  2. Add six times the pooled standard deviation to the mean obtained for each ELISA plate to obtain an ELISA positive threshold value. Any insect sample yielding an absorbance value equal to or greater than this value is deemed to be positive for the presence of the protein mark.

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Results

Internal marking:

The results of the internal mark retention test are shown in Figure 2A. The calculated ELISA critical threshold value was 0.054. Overall (all four sample dates combined), the insects treated without protein yielded consistently low ELISA values (x̄ = 0.038 ± 0.002, n = 80). Conversely, all of the insects fed the protein enriched diet yielded consistently strong ELISA v...

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Discussion

The arthropod protein immunomarking procedure was first described almost a quarter of a century ago9. Since then, the procedure has been adapted to study the dispersal patterns of a wide variety of arthropods using both internally and externally administered IgG/IgYs. These proteins have proven steadfast markers for the wide variety of insect species tested thus far. However, as mentioned above, the major limitation for using IgG/IgYs is that they are very expensive. Consequently, IgG/IgYs are only useful for ...

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Disclosures

Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

Acknowledgements

Funding was provided by USDA CRIS 5347-22620-021-00D and, in part, by the Agriculture and Food Research Initiative Competitive Grant no. 2011-67009-30141 from the USDA National Institute of Food and Agriculture. We are grateful for the technical support of Johanna Nassif. We also thank Paul Baker, David Horton, Diego Nieto, and Frances Sivakoff for providing some of the pictures used in Figure 3.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Food storage container (for insect rearing)Rubbermaid/TupperwareVarious sizesVarious manufacturers & vendors. Mesh fabric is glued in a window
Small volume nebulizer (Micro Mist)Teleflex-Hudson RCI1881Available online and in medical supply shops. Other brands will work as well.
Microcentrifuge tubes, 1.6 ml w/capContinental Lab Products4445.XAny similar type will work. We prefer brands that have easy to open lids.
Styrofoam storage box for microcentrifuge tubes (5x20 grid)RPI Corp.145746This design is nice because it doesn't crowd the tubes.
Dispenser, repeaterEppendorf4982000322Anything similar will help speed up the dispensing.
Pestles, plastic disposable tissue grinders, 1.5 mlKimble Chase749521-1500Available with various vendors. Pestles can be cleaned and autoclaved for reuse.
BD Falcon ELISA plates, 96-well (flat bottom)BD351172
Ovation pipette, manual (20-200 µl)VistaLab1057-0200 or 1070-0200Any similar type will work. This model is ergonomic.
Reservoirs, sterile reagentVistaLab4054-1000Anything similar will work.
Electronic pipette, 8-channel (50-1,200 µl) (Biohit eLine)Sartorius730391If you had to pick one electronic model, this size is most useful.
Electronic pipette, 8-channel (10-300 µl) (Biohit eLine)Sartorius730361Anything similar will work.
Manifold, multiwell plate washer (8-position, straight)Sigma-AldrichZ369802 Anything similar will work for manual plate washing.
Microplate reader with absorbance detectionMolecular DevicesSpectraMax 250Anything similar will work.
Chicken IgG/IgYUnited States BiologicalI1903-15RAlso available from other sources
Egg whitesGrocery store“All Whites”, “Egg Beaters”, etc.Mix 5 ml with 95 ml dH2O for 5% solution
Tris (Trizma Base)Sigma-AldrichT15032.42 g/L to make TBS
Sodium chloride (NaCl)Sigma-AldrichS988829.22 g/L to make TBS and 8.0 g⁠/⁠L for PBS
Sodium phosphate dibasic (Na2HPO4)Sigma-AldrichS08761.14 g/L for PBS
Potassium phosphate monobasic (KH2PO4)Sigma-AldrichP56550.2 g/L for PBS
Potassium chloride (KCl)Sigma-AldrichP95410.2 g/L for PBS
Tween 20, EIA gradeSigma-AldrichP1379Add 0.5 ml per L to PBS after salts are dissolved.
PBS with 1% BSASigma-AldrichP3688Mix 1 packet in 1 L dH2O
Anti-Chicken IgY (IgG) (whole molecule) antibody produced in rabbit (1:500)Sigma-AldrichC6409Mix 100 µl in 50 ml TBS
Dry Milk, Powdered or Fresh Milk (1%)Grocery storeMix 10 g with 1 L dH2O for 1% solution
Anti-Chicken IgY (IgG) (whole molecule)-Peroxidase antibody produced in rabbit (1:10,000)Sigma-AldrichA9046Mix 100 µl in 1 L of 1% milk
Anti-Chicken Egg Albumin antibody produced in rabbit (1:8,000)Sigma-AldrichC6534Dilute 125 µl in 1 L PBS-BSA
Goat Anti-Rabbit IgG Peroxidase Conjugate (1:2,000)Sigma-AldrichA6154Dilute 0.5 ml in 1 L PBS-BSA, then add 1.3 ml Silwet
Vac-In-Stuff (Silwet L-77) silicon-polyether copolymerLehle SeedsVIS-01Add as last ingredient
TMB Microwell One Component Peroxidase SubstrateSurModicsTMBW-1000-01

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Protein MarkingInsect DispersalELISA DetectionInternal TaggingExternal TaggingChicken IgYEgg WhiteSandwich ELISAIndirect ELISAMedical Nebulizer
Administering and Detecting Protein Marks on Arthropods for Dispersal Research
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