DOI: 10.1111/j.1570-7458.2010.00980.x A protein-based approach to mark arthropods for mark-capture type research James R. Hagler1* & Vincent P.Jones2 1USDA-ARS, Arid-Land Agricultural Research Center, 21881 North Cardon Lane, Maricopa, AZ 85138, USA, and 2Tree Fruit Research and Extension Center, Washington State University, 1100 N. Western Ave., Wenatachee, WA 98801, USA Accepted: 27 January 2010 Key words: arthropod marking, protein marking, ELISA, dispersal, cotton assemblage, Lygus hesperus, Hippodamia convergens, Trichoplusia ni, immunomarking, casein, egg albumin Abstract A series of studies was conducted to test methods for marking a wide variety of arthropods with inex- pensive proteins for mark-capture dispersal research. The markers tested included egg albumin pro- tein in chicken egg whites and casein protein in bovine milk. The first study qualified the effectiveness of the two marks on more than 50 arthropod species inhabiting cotton via two application proce- dures. The application methods included: (1) a topical plus residue protein application, and (2) a res- idue-only protein application. Both protein marks, regardless of the method of application, were readily retained on the arthropod assemblage over the duration of the study. The second study deter- mined how rapidly insects acquire chicken egg albumin protein after contact exposure to cotton tis- sue sprayed with an egg whites solution. Under laboratory conditions, the vast majority of adult Hippodamia convergens Gue´rin-Me´neville (Coleoptera: Coccinellidae) and Lygus hesperus Knight (Heteroptera: Miridae) acquired the mark after 5 min, and immature Trichoplusia ni (Hu¨bner) (Lepidoptera: Noctuidae) acquired the marker after 40 min. The third study determined how rapidly H. convergens and L. hesperus acquire bovine casein protein after contact exposure to either alfalfa, Medicago sativa L. (Fabaceae), or lesquerella, Lesquerella fendleri (Watson) (Brassicaceae), plants sprayed with a bovine milk solution. These insects rapidly acquired the casein mark from the plant residue under field conditions. A final study determined how long H. convergens retain casein protein after 24-h exposure to alfalfa and lesquerella plants containing a 7-day-old residue of bovine milk. Approximately 95% of the H. convergens maintained the casein mark for 2 days after removal from each type of plant. for any given study is strongly influenced by whether a Introduction mark-release-recapture (MRR) or mark-capture study is The success of quantifying arthropod dispersal patterns is needed. Of these, the mark-capture method is the most often dependent on having a reliable method to mark the difficult because the arthropods must be marked directly arthropod of interest. Over the years, researchers have used in the field. By process of elimination, the most useful many types of substances to mark arthropods (South- markers for mark-capture studies are materials that are wood, 1978; Hagler & Jackson, 2001; Lavandero et al., inexpensive and can be easily applied to vast areas directly 2004; Goubault & Hardy, 2007). The most suitable marker in the field by broadcast spray application. Paints, dyes, and dusts are the most common and least expensive types *Correspondence: James R. Hagler, USDA-ARS, Arid-Land Agricul- of arthropod markers available for MRR, but these are tural Research Center, 21881 North Cardon Lane, Maricopa, AZ usually not well suited for mark-capture type studies. 85138, USA. E-mail: [email protected] Rare or trace elements (e.g., rubidium, strontium, and Mention of proprietary or brand names are necessary to report factu- cesium) are arguably the most effective markers for mark- ally on available data; however, the USDA neither guarantees nor war- capture type studies (Berry et al., 1972; Stimmann et al., rants the standard of the product, and the use of the name by USDA implies no approval to the exclusion of others that also may be suit- 1973; Van Steenwyk et al., 1978; Fleischer et al., 1986; able. This article is in the public domain and not copyrightable. It Armes et al., 1989; Akey et al., 1991). Of these, rubidium may be freely reprinted with customary crediting of source. is by far the most frequently used trace element marker for No claim to US government works Entomologia Experimentalis et Applicata 135: 177–192, 2010 Journal compilation Ó 2010 The Netherlands Entomological Society 177 178 Hagler & Jones insects. Foliar applications of RbCl have been frequently monly used full season cotton, Gossypium hirsutum L. applied to various plant species to label a diverse array of (Malvaceae), cultivar ‘Delta Pine 5415’. The cotton was insects directly in the field (Frazer & Raworth, 1974; Stim- grown using standard agronomic practices. No insecticides mann, 1974; Shepard & Waddill, 1976; Graham et al., were applied to the field. This field site was purposefully 1978a,b; Alverson et al., 1980; Corbett et al., 1996; Tillman selected for this study because it contained a relatively low et al., 2007). However, analytical methods to determine population of arthropods, due to it being surrounded by a presence of trace elements in insects are relatively costly, large industrial park within the Phoenix metropolitan area. time consuming, and requires technical expertise (Akey This feature allowed us to manipulate, to a certain degree, et al., 1991; Hagler & Jackson, 2001). the arthropod populations introduced into each of the A major breakthrough for marking insects for dispersal field cage treatments. Field cages (1.83 · 1.83 · 1.83 m) studies was the development of the so-called protein mark- were erected in randomly located areas within the study ing or immunomarking procedure. The method consists site and the roughly 90 cotton plants enclosed in each cage of marking arthropods with an exogenous protein. In turn, were sprayed with one of the two proteins described the protein marker is detected in or on the arthropods by below. an anti-protein-specific enzyme-linked immunosorbent assay (ELISA). The first generation of protein markers Test arthropods. We augmented the arthropod population included rabbit IgG and chicken IgG proteins (Hagler in each field cage to insure that we had enough arthropods et al., 1992; Hagler, 1997). These proteins have been effec- to conduct a lengthy study. Arthropods were collected en tive for marking a wide variety of insects for MRR-type masse on 29 June for the chicken egg white study, and 12 research (Hagler et al., 1992, 2002; DeGrandi-Hoffman & August 2004 for the bovine milk study, using standard 38- Hagler, 2000; Blackmer et al., 2004; Hagler & Naranjo, cm diameter sweepnets from densely populated cotton 2004; Peck & McQuate, 2004; Buczkowski & Bennett, and alfalfa fields located at the University of Arizona Mari- 2006; Jasrotia & Ben-Yakir, 2006; Janke et al., 2009). How- copa Agricultural Center, Maricopa, AZ. The arthropods ever, the major drawback with IgG protein marks is that were placed into large paper bags, sealed, and transported they are too expensive for mark-capture studies. Recently, to the study site. Half of the sweepnet samples were second generation protein-specific ELISAs were developed released into two of four field cages on the same day they to detect egg albumin protein contained in chicken egg were collected for the topical plus residual mark applica- whites, soy protein in soy milk, and casein protein tion treatment (see below). The other half of the bagged contained in bovine milk. Each ELISA is highly sensitive arthropod samples were stored overnight in a refrigerator and specific to its targeted protein and, most importantly, (4 °C) and then released the next day into the remaining the proteins are readily available at only a fraction of the two field cages for the residual mark-only application cost of IgG proteins (Jones et al., 2006). treatment (see below). In this article, we evaluate various aspects of the acquisi- tion and persistence of casein and egg albumin markers on Protein markers. Two protein-rich materials, chicken egg a wide variety of arthropods under laboratory and field whites (All WhitesÔ; Papetti Foods, Elizabeth, NJ, USA) conditions. We also discuss the potential advantages and and non-fat dry milk (Kroger, Cincinnati, OH, USA) were limitations of the protein marking procedure for marking evaluated as potential marks for the arthropod complex arthropods for large-scale mark-capture type dispersal introduced into each cage. studies. Chicken egg whites evaluation: Half of the sweepnet samples were released in equal numbers into two ran- domly selected field cages described above on 29 June Materials and methods 2004. The arthropods were given a 24-h acclimation First study – Acquisition of protein marks by an arthropod assem- period to settle in the cages before application of 1 l of a blage 10% (vol ⁄ vol) solution of chicken egg whites on 30 June Study site. Field cage studies were conducted to determine using a 17-l backpack sprayer (Spray DocÔ; Gilmour the acquisition of egg albumin and bovine casein protein Gardening Innovation, Gilmour, Somerset, PA, USA). marks by an assemblage of arthropods. Each protein was Four hours later, after the spray solution had dried (note: administered to the arthropod complex using two differ- the temperature was ca. 40 °C), the other half of the ent approaches. The field cages were erected during the arthropod sweepnet samples collected the day before was summer of 2004, at a 1.2-ha field site located at the released into the two remaining cages. This design resulted USDA-ARS, Western Cotton Research Laboratory, in arthropods in the first two cages potentially picking up Phoenix, AZ, USA. The field was planted with the com- the marker via either direct contact with the spray solution Marking arthropods for mark-capture studies 179 or by residual contact after the marker solution dried; between samples. Arthropod and cotton leaf tissue samples those in the latter two cages could only acquire the mark were collected from each cage every other day until all the by residual contact.