Laboratory investigation of triple marking the parasitoid Gonatocerus ashmeadi with a fluorescent dye and two animal proteins

Gonatocerus ashmeadi Girault (Hymenoptera: Mymaridae), a parasitoid of Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) (glassy‐winged sharpshooter), was used as a model insect to investigate triple marking a minute hymenopteran for potential use for monitoring dispersal patterns of natural enemies in the field. The triple mark contained egg albumin in chicken eggs, casein in bovine milk, and SARDI yellow fluorescent dye. Three application treatments of the triple mark were investigated: (1) a wet topical treatment, (2) a dry residue treatment, and (3) untreated control. The presence of albumin and casein protein marks were detected by an anti‐albumin and anti‐casein enzyme‐linked immunosorbent assays (ELISA) using both ‘soaked’ and ‘crushed’ specimens. Of the topically treated parasitoids, yellow dye, casein, and albumin were detected on 88, 69, and 21% of the crushed samples, respectively. The yellow dye and casein (tested with crush ELISA) were the most efficient marking methods, detecting up to 29% more marked G. ashmeadi. Yellow dye resulted in zero false positives in the untreated control. The percentage of false positives for casein (tested with crush ELISA) was 1.3; however, this was reduced to 0% when a double‐marking system using any two of the three marks (yellow dye, casein, and albumin) were used to mark parasitoids. This double‐mark system resulted in 65% of parasitoids being successfully marked in the topical treatment over the duration of the study. For casein, crush ELISA was 26% more sensitive and 24% more accurate than soak ELISA for detecting this mark. Yellow dye, albumin, and casein (tested with crush ELISA) were retained on marked parasitoids for the entire duration of the 11‐day study. Parasitoids self‐marked with yellow dye, albumin (tested with soak ELISA), casein (tested with crush ELISA), and the double‐mark (tested with crush ELISA) by walking over dried residue of the triple mark. This resulted in up to 17% more marked parasitoids in the residue treatment compared with the untreated control. A topical application of the triple mark had no effect on survival of G. ashmeadi compared with the control. The residue treatment resulted in significantly lower mortality than the untreated control, indicating that G. ashmeadi may have fed on the protein in the residue of the triple‐mark, which enhanced longevity.