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.

1. Internal Mark, Retention, and Detection Procedure
<ol>
	<li>Internal marking procedure
	<ol>
		<li>Collect insects of interest (n &#8776; 100 individuals) from a laboratory colony reared on an artificial diet or from the field and divide into two clean rearing containers.</li>
		<li>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).</li>
		<li>Allow the insects to resume their feeding activity for 24 hr.</li>
	</ol>
	</li>
	<li>Internal mark retention procedure
	<ol>
		<li>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).</li>
		<li>Collect a cohort (n = 20) of insects daily (or at any desired time interval) from each treatment and freeze immediately at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Sandwich ELISA procedure
	<ol>
		<li>Add 50 &#181;l 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 &#176;C).</li>
		<li>Discard the antibody from the plate and block each well with 300 &#181;l of a 1.0% non-fat dry milk solution for 30 min.</li>
		<li>While waiting for the previous incubation steps, place frozen insects individually in 1.6 ml microcentrifuge tubes with 1.0 ml of TBS.</li>
		<li>Grind each insect with a clean pestle until thoroughly homogenized and add a 100 &#181;l of each sample to an individual well of the ELISA plate. Allow the samples to incubate at RT for 1 hr.</li>
		<li>Add 100 &#181;l of negative (TBS only) and positive (egg whites in TBS) controls to wells in the first column of each ELISA plate. Also, add 100 &#181;l 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.</li>
		<li>Wash each well three times with phosphate buffered saline (PBS)-tween and then add 50 &#181;l 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.</li>
		<li>Wash each well three times with PBS-tween and add 50 &#181;l of tetramethylbenzidine (TMB) substrate to each well.</li>
		<li>After 10 min, read the wells with a microplate spectrophotometer set at a wavelength of 650 nm.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53693/53693fig1.jpg" /> 
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&#8230;80), and 8 wells dedicated to unmarked (negative) insect controls (Neg Ctrl 1&#8230;8). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53693/53693fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. External Mark, Retention, and Detection Procedure
<ol>
	<li>External marking procedure
	<ol>
		<li>Collect insects of interest (n &#8776; 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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Continue spraying (marking) until the egg whites solution has been dispensed (approx. 2-5 min).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>External mark retention procedure
	<ol>
		<li>Place each treatment of the dry insects into a separate clean rearing container to avoid further exposure to the mark.</li>
		<li>Add food and water to the clean containers to sustain the insects over the duration of the study.</li>
		<li>Collect a cohort (n = 20) of insects from each treatment daily and freeze immediately at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Indirect ELISA procedure
	<ol>
		<li>Place the frozen insects individually into 1.6 ml microcentrifuge tubes and add 1.0 ml of TBS.</li>
		<li>Soak the samples for 1 hr on an orbital shaker set at 120 rpm.</li>
		<li>Add negative and positive controls as described in Step 1.3.5.</li>
		<li>After soaking, pipette a 100 &#181;l 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 &#176;C).</li>
		<li>Wash the wells three times with PBS-tween and then add 300 &#181;l of phosphate buffered saline containing 1% bovine serum albumin (PBS-BSA) to each well.</li>
		<li>After 30 min at RT wash twice with PBS-tween.</li>
		<li>Add 50 &#181;l 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.</li>
		<li>Wash each well three times with PBS-tween and then add 50 &#181;l of goat anti-rabbit IgG conjugated with horseradish peroxidase diluted 1:2,000 in PBS-BSA-Silwet (0.05%) for 1 hr at RT.</li>
		<li>Wash each well three times with PBS-tween and add 50 &#181;l of TMB substrate to each well.</li>
		<li>After 10 min, read the wells with a microplate spectrophotometer set at a wavelength of 650 nm.</li>
	</ol>
	</li>
</ol>
3. Data Analysis&#160;
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>

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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 MRR and self-marking type studies where large batches of insects can be marked in a small space or the marks can be incorporated into a diet or bait. Here, we provided a simple demonstration of how an internal mark can be administered by feeding the target insect an artificial diet enriched with chicken IgG/IgY. In turn, the IgG/IgY was detected using a sandwich ELISA. This system might be especially advantageous to researchers that already use a diet that contains chicken eggs since the assay will naturally detect the IgG/IgY found in eggs. In addition, small amounts of IgG/IgY protein can be administered topically to insects using the medical nebulizer described above (Figure 3A). A nebulizer was first used to mass mark very delicate and tiny parasitoids with rabbit IgG for MRR type research3. Since then, other methods have been used to administer IgG/IgY marks to arthropods for dispersal research. For example, a perfume atomizer has been used to apply IgG/IgY marks on other parasitoid species13,14. Others have internally marked various insects by feeding them (i.e., self-marking) rabbit IgG labeled honey14,15 or sugar water16 (Figure 3B). Finally, Baker et al.7 fed cardboard to termites that was impregnated with rabbit IgG (Figure 3C). These termites readily self-marked as they ingested the baited foodstuffs.
<img alt="Figure 3" src="/files/ftp_upload/53693/53693fig3.jpg" /> 
Figure 3. Various methods used to administer a protein mark: (A) delicate parasitoids being marked with chicken IgY using a medical nebulizer, (B) a parasitoid feeding on rabbit IgG-enriched honey solution, (C) termites feeding on a rabbit IgG-enriched cardboard bait, (D) honey bees self-marking with dry milk as they go through a dusting device placed at the entrance of a hive, (E) native insects being marked with milk in a cotton field using a conventional tractor spray rig, (F) native insects being marked with chicken egg whites with a boom and nozzle spray device mounted onto an ATV, (G) native insects in a strip of alfalfa being marked with chicken egg whites with a backpack spray device, and (H,&#160;I) native insects in alfalfa being marked with chicken egg whites using aerial applicators. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53693/53693fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
IgG/IgY proteins have not just been used for studying insect dispersal. They have also been used for tracking trophic links in the arthropod food chain. Hagler and Durand17 first proposed the concept of marking prey with IgG/IgY and, in turn, conducting an IgG/IgY-specific sandwich gut content ELISA on predators that consumed the marked prey. This technique has recently gained popularity among gut analysis researchers because it is less tedious and costly than conducting prey-specific ELISA or polymerase chain reaction (PCR) type molecular gut content analyses (see Hagler18 and Fournier et al.19 for reviews of the pros and cons of the various gut assay techniques). Within the past decade, the prey immunomarking technique has been used to identify feeding activity of cotton predators on rabbit IgG-marked pests18,20,21, trophallaxis and feeding relationships in termite colonies using a rabbit IgG-treated paper food source22, granivory of an arthropod assemblage on invasive dandelion weed seeds marked with rabbit IgG23, stink bug predation rates on IgG-marked tomato hornworms collected in plots subjected to various semiochemical treatments24, and predator scavenging activity on IgG/IgY-marked carrion25.
A second generation of protein detection indirect ELISAs were developed almost a decade ago. These ELISAs were designed specifically to detect proteins contained in &#34;off-the-shelf&#34; food products, such as egg albumin protein in chicken egg whites, soy trypsin inhibitor protein in soy milk, and casein protein in bovine milk. The second-generation protein immunomarkers have proven useful for MRR, mark-capture and self-mark type research. For instance, Irvin et al.26 showed that it was feasible to topically mark delicate parasitoids with these off-the-shelf proteins. Moreover, Hagler et al.8,27 showed that honey bees can self-mark with egg white and bovine milk dry powders as they exit through a protein dispenser placed at the entrance of a hive (Figure 3D). Perhaps the most creative use of the protein marking involved targeting cow pats with egg whites using a jet sprayer28. In turn, face fly adults acquired a self-mark as they emerged from their pupal stage and exited the cow pat.
The most important characteristic of the second-generation protein immunomarkers is that they are readily available in mass quantities and at a fraction of the cost of IgG/IgY proteins11. This feature has revolutionized the way area-wide mark-capture type research is conducted. Specifically, investigators now have a reliable method to rapidly and uniformly tag arthropods over vast areas in the field using a wide variety of conventional spray rig equipment (Figure 3E). For instance, pest insects have been marked directly in orchards and fields using portable hand gun type sprayers29,30, tractor-driven airblast sprayers31,32, and skid sprayers33. Horton et al.34 used an electric spray device mounted on an all-terrain vehicle to pinpoint applications of egg whites to insects occupying cover crops embedded in a pear orchard (Figure 3F). Similarly, Swezey et al.5,35 used a backpack sprayer to apply precise applications of chicken egg whites to rows of an alfalfa trap crop (a preferred host plant) embedded in an organically grown strawberry field to mark a pest and its natural enemies (Figure 3G). The results from that research showed that the pest and natural enemies were retained by the alfalfa trap crop and rarely dispersed into the highly valued strawberries. Perhaps the most extreme application of a protein immunomarker was through the use of an aerial helicopter and airplane crop dusters. In that study, Sivakoff et al.36 applied a broadcast application of bovine milk and egg white to 29 and 16 ha of blooming alfalfa, respectively (Figure 3H, I). The aim of that study was to mark the resident arthropods in the alfalfa, and then, after the alfalfa was harvested by mowing (sparking a dispersal event), to track the dispersal patterns of the arthropods into adjacent cotton fields (a crop susceptible to the pest species).
Some users of the second-generation mark-capture procedure have modified the original procedure somewhat to meet their specific research needs. The body type and size of the targeted insect, along with its behavioral characteristics, will dictate the volume and concentration of mark needed and how the mark is administered37. Moreover, the method used to recapture insects in the field will be contingent on these factors. To date, almost every method used (e.g., sticky traps, sweep nets, vacuums) to collect insects has been used successfully to collect marked insects3,5,8,11,29,31,32,35,38. However, due to the extreme sensitivities of the protein detection ELISAs, we emphasize that great care must be taken to ensure that unmarked specimens do not become contaminated during the collection or sorting processes38.
The original study11 that described the second generation of protein marking systems (i.e., egg albumin, milk casein, and soy trypsin inhibitor) reported factors affecting assay sensitivity may vary among the types of marker. Specifically, they showed that different water sources, additives (surfactants), and even the type of ELISA plate used affected (either positively or negatively) the assay. Moreover, that study and subsequent studies37,39,40 showed that the three protein mark detection assays vary in efficacy. The egg white marker was retained longer than the milk marker, which was retained longer than soy milk marker. These results highlight the importance of testing protein mark retention on any given insect prior to embarking on field research. Also, these second-generation indirect ELISAs sometimes show a low level of background noise with certain types of insects (e.g., herbivores) (pers. obs). This background noise can be reduced by adding 100 &#181;l of hydrogen peroxide to each well of the ELISA plate for 30 min&#160;between Step 2.3.4 and Step 2.3.5 in the protocol above (unpubl. data).
It should be noted that the indirect ELISAs used to detect the second-generation protein marks are very effective at detecting the protein on insects soaked in the sample buffer. However, the indirect ELISAs are not as effective as the sandwich ELISA at detecting internal protein marks or prey proteins on insects. Studies have shown that indirect ELISAs are not as efficient as sandwich ELISAs at detecting protein in homogenized insect samples34,41.
An important factor to consider with any ELISA is that the procedure often shows plate-to-plate (attributed mainly to day-to-day effects of running an assay) variability42,43. Therefore, it is essential that every ELISA plate includes: (1) one or more TBS only negative controls, (2) one or more TBS + pure protein positive controls, and (3) at least eight negative insect controls (i.e., individuals known not to contain a protein mark). The TBS only, TBS + protein, and insect negative controls assure that the buffer is not a source of error, the reagents are working properly, and that there are not any proteins in the insect that cross react with the ELISA reagents, respectively. Moreover, the insect negative controls are essential for calculating ELISA threshold values (see below). The plate design shown in this demonstration included 7 TBS blanks, 1 protein + TBS positive control (in the first column) and eight negative controls (in the last column) on each ELISA plate (Figure 1).
Another important factor to consider when handling ELISA data is to determine a threshold value to score an insect for the presence or absence of a protein mark. The method originally proposed by Stimmann44 for scoring rubidium-marked insects was defined as the mean level of marker in a pool of unmarked insects plus three times the standard deviation of the unmarked individuals. This method has also been used as the conventional method for scoring insects for the presence of protein marks9,17. However, in some cases, researchers have intuitively selected more conservative threshold values to reduce the likelihood of obtaining false positives. The easiest method is to just add four to six standard deviations to the mean of the negative controls instead of three18,34. A more sophisticated method to further reduce the chance of obtaining false positives was proposed by Sivakoff et al.12. This method entails calculating the mean absorbance value of the eight negative control insects on each ELISA plate and then calculating the standard deviation based on the pooled negative controls from all of the ELISA plates of a given study.
In summary, a reliable method to tag arthropods is critical to the success of most dispersal studies. Various methods have been used to mark arthropods with proteins over the past two decades. Protein immunomarking and the detection of the marks are relatively easy and inexpensive and very adaptable to a myriad of studies. Future users of the immunomarking technique should ensure that their methodology is suitable to the specifics of their research. The concentration and volume of the mark needed and how the mark is applied will depend on factors like behavior, body type and size of the target species40.

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&#39;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 &#34;brushing&#34; 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&#39;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.

<table>
	<tbody>
		<tr>
			<td>Food storage container (for insect rearing)</td>
			<td>Rubbermaid/Tupperware</td>
			<td>Various sizes</td>
			<td>Various manufacturers &#38; vendors. Mesh fabric is glued in a window `</td>
		</tr>
		<tr>
			<td>Small volume nebulizer (Micro Mist)</td>
			<td>Teleflex-Hudson RCI</td>
			<td>1881</td>
			<td>Available online and in medical supply shops. Other brands will work as well.</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, 1.6 ml w/cap</td>
			<td>Continental Lab Products</td>
			<td>4445.X</td>
			<td>Any similar type will work. We prefer brands that have easy to open lids.</td>
		</tr>
		<tr>
			<td>Styrofoam storage box for microcentrifuge tubes (5x20 grid)</td>
			<td>RPI Corp.</td>
			<td>145746</td>
			<td>This design is nice because it doesn&#39;t crowd the tubes.</td>
		</tr>
		<tr>
			<td>Dispenser, repeater</td>
			<td>Eppendorf</td>
			<td>4982000322</td>
			<td>Anything similar will help speed up the dispensing.</td>
		</tr>
		<tr>
			<td>Pestles, plastic disposable tissue grinders, 1.5 mL</td>
			<td>Kimble Chase</td>
			<td>749521-1500</td>
			<td>Available with various vendors. Pestles can be cleaned and autoclaved for reuse.</td>
		</tr>
		<tr>
			<td>BD Falcon ELISA plates, 96-well (flat bottom)</td>
			<td>BD</td>
			<td>351172</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovation pipette, manual (20-200 &#181;l)</td>
			<td>VistaLab</td>
			<td>1057-0200 or 1070-0200</td>
			<td>Any similar type will work. This model is ergonomic.</td>
		</tr>
		<tr>
			<td>Reservoirs, sterile reagent</td>
			<td>VistaLab</td>
			<td>4054-1000</td>
			<td>Anything similar will work.</td>
		</tr>
		<tr>
			<td>Electronic pipette, 8-channel (50-1200 &#181;l) (Biohit eLine)</td>
			<td>Sartorius</td>
			<td>730391</td>
			<td>If you had to pick one electronic model, this size is most useful.</td>
		</tr>
		<tr>
			<td>Electronic pipette, 8-channel (10-300 &#181;l) (Biohit eLine)</td>
			<td>Sartorius</td>
			<td>730361</td>
			<td>Anything similar will work.</td>
		</tr>
		<tr>
			<td>Manifold, multiwell plate washer (8-position, straight)</td>
			<td>Sigma-Aldrich</td>
			<td>Z369802&#160;</td>
			<td>Anything similar will work for manual plate washing.</td>
		</tr>
		<tr>
			<td>Microplate reader with absorbance detection</td>
			<td>Molecular Devices</td>
			<td>SpectraMax 250</td>
			<td>Anything similar will work.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken IgG/IgY</td>
			<td>United States Biological</td>
			<td>I1903-15R</td>
			<td>Also available from other sources</td>
		</tr>
		<tr>
			<td>Egg whites</td>
			<td>Grocery store</td>
			<td>&#8220;All Whites&#8221;, &#8220;Egg Beaters&#8221;, etc.</td>
			<td>Mix 5 mL with 95 mL dH2O for 5% solution</td>
		</tr>
		<tr>
			<td>Tris (Trizma Base)</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td>2.42 g/L to make TBS</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>29.22 g/L to make TBS and 8.0 g/L for PBS</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S0876&#160;</td>
			<td>1.14 g/L for PBS</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>0.2 g/L for PBS</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>0.2 g/L for PBS</td>
		</tr>
		<tr>
			<td>Tween 20, EIA grade</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td>Add 0.5 mL per L to PBS after salts are dissolved.</td>
		</tr>
		<tr>
			<td>PBS with 1% BSA</td>
			<td>Sigma-Aldrich</td>
			<td>P3688</td>
			<td>Mix 1 packet in 1 L dH20</td>
		</tr>
		<tr>
			<td>Anti-Chicken IgY (IgG) (whole molecule) antibody produced in rabbit (1:500)</td>
			<td>Sigma-Aldrich</td>
			<td>C6409</td>
			<td>Mix 100 &#181;l in 50 ml TBS</td>
		</tr>
		<tr>
			<td>Dry Milk, Powdered or Fresh Milk (1%)</td>
			<td>Grocery store</td>
			<td></td>
			<td>Mix 10 g with 1 L dH2O for 1% solution</td>
		</tr>
		<tr>
			<td>Anti-Chicken IgY (IgG) (whole molecule)-Peroxidase antibody produced in rabbit (1:10000)</td>
			<td>Sigma-Aldrich</td>
			<td>A9046</td>
			<td>Mix 100 ul in 1 L of 1% milk</td>
		</tr>
		<tr>
			<td>Anti-Chicken Egg Albumin antibody produced in rabbit (1:8000)</td>
			<td>Sigma-Aldrich</td>
			<td>C6534</td>
			<td>Dilute 125 &#181;l in 1 L PBS-BSA</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG Peroxidase Conjugate (1:2000)</td>
			<td>Sigma-Aldrich</td>
			<td>A6154</td>
			<td>Dilute 0.5 ml in 1 L PBS-BSA, then add 1.3 ml Silwet</td>
		</tr>
		<tr>
			<td>Vac-In-Stuff (Silwet L-77)silicon-polyether copolymer</td>
			<td>Lehle Seeds</td>
			<td>VIS-01</td>
			<td>Add as last ingredient</td>
		</tr>
		<tr>
			<td>TMB Microwell One Component Peroxidase Substrate</td>
			<td>SurModics</td>
			<td>TMBW-1000-01</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

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 (x&#772; = 0.038 &#177; 0.002, n = 80). Conversely, all of the insects fed the protein enriched diet yielded consistently strong ELISA values (x&#772; = 0.475 &#177; 0.221, n = 80).
External marking:
The results of the external mark retention test are shown in Figure 2B. The calculated ELISA critical threshold value was 0.082. Overall, the insects topically marked with only water yielded consistently low ELISA values (x&#772; = 0.048 &#177; 0.007, n = 80). Conversely, all of the insects topically marked with the egg whites solution yielded consistently strong ELISA values (x&#772;= 0.746 &#177; 0.232, n = 80).
<img alt="Figure 2" src="/files/ftp_upload/53693/53693fig2.jpg" /> 
Figure 2. Mean (&#177; SD) ELISA optical density values of (A) insects marked internally with chicken IgY and (B) insects marked externally with chicken egg whites (albumin). Black bars are the mean &#177; SD ELISA optical density values yielded by unmarked insects. Insect sample size for each daily protein treatment was 20 individuals. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53693/53693fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Administering and Detecting Protein Marks on Arthropods for Dispersal Research at JoVE.com

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

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 ...

administering-detecting-protein-marks-on-arthropods-for-dispersal

Nanyang Technological University

Research

JoVE Journal

Environment

7.3K Views.  Arid-Land Agricultural Research Center. 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. 

Video: Administering and Detecting Protein Marks on Arthropods for Dispersal Research

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 ...

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 ...

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

RGB and Spectral Root Imaging for Plant Phenotyping and Physiological Research: Experimental Setup and Imaging Protocols

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop cultivars against environmental stresses. An experimental protocol is presented for RGB and hyperspectral imaging of root systems. The approach uses rhizoboxes where plants grow in natural soil over a longer time to observe fully developed root systems. Experimental settings are exemplified for assessing rhizobox plants under water stress and studying the role of roots. An RGB imaging setup is described for cheap and quick quantification of root development over time. Hyperspectral imaging improves root segmentation from the soil background compared to RGB color based thresholding. The particular strength of hyperspectral imaging is the acquisition of chemometric information on the root-soil system for functional understanding. This is demonstrated with high resolution water content mapping. Spectral imaging however is more complex in image acquisition, processing and analysis compared to the RGB approach. A combination of both methods can optimize a comprehensive assessment of the root system. Application examples integrating root and aboveground traits are given for the context of plant phenotyping and plant physiological research. Further improvement of root imaging can be obtained by optimizing RGB image quality with better illumination using different light sources and by extension of image analysis methods to infer on root zone properties from spectral data.

An experimental protocol is presented for assessment of soil grown plant root systems with RGB and hyperspectral imaging. Combination of RGB image time series with chemometric information from hyperspectral scans optimizes insights into plant root dynamics.

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop ...

Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive species known to redox regulate biological processes in health and disease. Pre-clinical and clinical evidence points to beneficial effects of plasma treatment in the healing of chronic ulcer of the skin. Other emerging topics, such as plasma cancer treatment, are receiving increasing attention. Plasma medical research requires interdisciplinary expertise in physics, chemistry, and biomedicine. One goal of plasma research is to characterize plasma-treated cells in a variety of specific applications. This includes, for example, cell count and viability, cellular oxidation, mitochondrial activity, cytotoxicity and mode of cell death, cell cycle analysis, cell surface marker expression, and cytokine release. This study describes the essential equipment and workflows required for such research in plasma biomedicine. It describes the proper operation of an atmospheric pressure argon plasma jet, specifically monitoring its basic emission spectra and feed gas settings to modulate reactive species output. Using a high-precision xyz-table and computer software, the jet is hovered in millisecond-precision over the cavities of 96-well plates in micrometer-precision for maximal reproducibility. Downstream assays for liquid analysis of redox-active molecules are shown, and target cells are plasma-treated. Specifically, melanoma cells are analyzed in an efficient sequence of different consecutive assays but using the same cells: measurement of metabolic activity, total cell area, and surface marker expression of calreticulin, a molecule important for the immunogenic cell death of cancer cells. These assays retrieve content-rich biological information about plasma effects from a single plate. Altogether, this study describes the essential steps and protocols for plasma medical research.

A high-throughput cold physical plasma treatment protocol of liquids and cells is shown. It involves setting up different feed gas compositions for plasma ignition, measuring the plasma&#39;s emission spectra, and the subsequent analysis of liquids and cellular activity after plasma treatment.

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive ...

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 ...

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

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 ...

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are also a useful scientific model to study motion vision and flight dynamics in a controlled laboratory setting. As the larvae develop in organically polluted water, they are useful models for investigating investment in microbial immunity. While large scale commercial breeding for agriculture already occurs, there are no standardized protocols for maintaining captive populations for scientific studies. This is important as commercial captive breeding programs focusing on mass output during peak pollination periods may fail to provide a population that is consistent, stable and robust throughout the year, as is often needed for other research purposes. Therefore, a method to establish, maintain and refresh a captive research population is required. Here, we describe the utilization of an artificial hibernation cycle, in addition to the nutritional and housing requirements, for long term maintenance of Eristalis tenax. Using these methods, we have significantly increased the health and longevity of captive populations of E. tenax compared to previous reports. We furthermore discuss small scale rearing methods and options for optimizing yields and manipulating population demographics.

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

The overall goal of these procedures is to establish, maintain and refresh a captive population of Eristalis tenax in a research setting.

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are ...