Administering and Detecting Protein Marks on Arthropods for Dispersal Research

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 species^13,14. Others have internally marked various insects by feeding them (i.e., self-marking) rabbit IgG labeled honey^14,15 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.

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, I) native insects in alfalfa being marked with chicken egg whites using aerial applicators. Please click here to view a larger version of this figure.

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 pests^18,20,21, 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 "off-the-shelf" 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 sprayers^29,30, tractor-driven airblast sprayers^31,32, 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 insects^{3,5,8,11,29,31,32,35,38}. 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 studies^37,39,40 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 µl of hydrogen peroxide to each well of the ELISA plate for 30 min 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 samples^34,41.

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) variability^42,43. 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 marks^9,17. 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 three^18,34. 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.