Administering and Detecting Protein Marks on Arthropods for Dispersal Research

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 interactions^1,2.

The appropriateness of a marker used for any given dispersal research will be de....

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 no....

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.......

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Food storage container (for insect rearing)	Rubbermaid/Tupperware	Various sizes	Various manufacturers & vendors. Mesh fabric is glued in a window
Small volume nebulizer (Micro Mist)	Teleflex-Hudson RCI	1881	Available online and in medical supply shops. Other brands will work as well.
Microcentrifuge tubes, 1.6 ml w/cap	Continental Lab Products	4445.X	Any similar type will work. We prefer brands that have easy to open lids.
Styrofoam storage box for microcentrifuge tubes (5x20 grid)	RPI Corp.	145746	This design is nice because it doesn't crowd the tubes.
Dispenser, repeater	Eppendorf	4982000322	Anything similar will help speed up the dispensing.
Pestles, plastic disposable tissue grinders, 1.5 ml	Kimble Chase	749521-1500	Available with various vendors. Pestles can be cleaned and autoclaved for reuse.
BD Falcon ELISA plates, 96-well (flat bottom)	BD	351172
Ovation pipette, manual (20-200 µl)	VistaLab	1057-0200 or 1070-0200	Any similar type will work. This model is ergonomic.
Reservoirs, sterile reagent	VistaLab	4054-1000	Anything similar will work.
Electronic pipette, 8-channel (50-1,200 µl) (Biohit eLine)	Sartorius	730391	If you had to pick one electronic model, this size is most useful.
Electronic pipette, 8-channel (10-300 µl) (Biohit eLine)	Sartorius	730361	Anything similar will work.
Manifold, multiwell plate washer (8-position, straight)	Sigma-Aldrich	Z369802	Anything similar will work for manual plate washing.
Microplate reader with absorbance detection	Molecular Devices	SpectraMax 250	Anything similar will work.
Chicken IgG/IgY	United States Biological	I1903-15R	Also available from other sources
Egg whites	Grocery store	“All Whites”, “Egg Beaters”, etc.	Mix 5 ml with 95 ml dH2O for 5% solution
Tris (Trizma Base)	Sigma-Aldrich	T1503	2.42 g/L to make TBS
Sodium chloride (NaCl)	Sigma-Aldrich	S9888	29.22 g/L to make TBS and 8.0 g⁠/⁠L for PBS
Sodium phosphate dibasic (Na2HPO4)	Sigma-Aldrich	S0876	1.14 g/L for PBS
Potassium phosphate monobasic (KH2PO4)	Sigma-Aldrich	P5655	0.2 g/L for PBS
Potassium chloride (KCl)	Sigma-Aldrich	P9541	0.2 g/L for PBS
Tween 20, EIA grade	Sigma-Aldrich	P1379	Add 0.5 ml per L to PBS after salts are dissolved.
PBS with 1% BSA	Sigma-Aldrich	P3688	Mix 1 packet in 1 L dH2O
Anti-Chicken IgY (IgG) (whole molecule) antibody produced in rabbit (1:500)	Sigma-Aldrich	C6409	Mix 100 µl in 50 ml TBS
Dry Milk, Powdered or Fresh Milk (1%)	Grocery store		Mix 10 g with 1 L dH2O for 1% solution
Anti-Chicken IgY (IgG) (whole molecule)-Peroxidase antibody produced in rabbit (1:10,000)	Sigma-Aldrich	A9046	Mix 100 µl in 1 L of 1% milk
Anti-Chicken Egg Albumin antibody produced in rabbit (1:8,000)	Sigma-Aldrich	C6534	Dilute 125 µl in 1 L PBS-BSA
Goat Anti-Rabbit IgG Peroxidase Conjugate (1:2,000)	Sigma-Aldrich	A6154	Dilute 0.5 ml in 1 L PBS-BSA, then add 1.3 ml Silwet
Vac-In-Stuff (Silwet L-77) silicon-polyether copolymer	Lehle Seeds	VIS-01	Add as last ingredient
TMB Microwell One Component Peroxidase Substrate	SurModics	TMBW-1000-01