Evaluation of water-soluble dyes to mark internal structures of Lepidoptera via larval feeding

ABSTRACT Understanding aspects of insect ecology is a key component for the applicability of control methods or pest resistance management. For instance, the comprehension of insect dispersal is crucial to determining insect gene flow and the maximum distance between refuge areas in Bt (Bacillus thuringiensis) crops. But, for such studies, insects need to be marked prior to release. Seeking to refine the technique of dispersion studies of the genetic material of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), this study aimed to determine the efficiency of water-soluble dyes for the internal marking of S. frugiperda immatures and adults via larval feeding, with emphasis on marking the spermatophore. Rhodamine B, Methylene Blue, Ponceau, Coomassie, and Eosin-Nigrosin were added to the larval artificial diet at concentrations of 0, 100, 200, 400, 600, 800, and 1000 ppm. Mortality and duration of the larval stage as well as the efficiency of marking internal structures of larvae and adults were evaluated. Rhodamine B and Methylene Blue increased the duration of the larval period, but the former caused higher mortality. The staining of the gut, hemolymph, and imaginal discs, in the larval phase, was observed for Rhodamine B, Methylene Blue, and Coomassie dyes. However, none of the dyes were efficient for marking spermatophores. Thus, the addition of these water-soluble dyes to the larval diet of S. frugiperda can mark internal structures of the larva, but not the adult. Therefore, these dyes have limited application once they may be used only for marking larvae.

Immigration of Lygus spp. (Hemiptera: Miridae) and predaceous natural enemies to trap-cropped organic strawberry.

Lygus spp. are polyphagous pests that overwinter in weedy vegetation. In the spring on the central coast of California, Lygus spp. emigrate from weeds into strawberry fields. Subsequent feeding on strawberry flowers causes fruit deformation that precludes sale on the fresh market. Use of alfalfa (Medicago sativa L.) (Fabales: Fabaceae) as a trap crop has been hypothesized to prevent Lygus spp. colonization in strawberries. We examined the movement of Lygus spp. and associated predators from weeds to strawberry fields with alfalfa trap crops using a protein mark-capture technique. Insects and spiders were collected from weeds, strawberry, and alfalfa 1 day, 2 days, and ~2 wk after an albumin protein mark was applied to weeds bordering strawberry fields. For marked Lygus spp. that emigrated from weeds, the majority (79%) of adults were recovered from alfalfa trap crops; however, all nymphs immigrated to strawberry. Most protein-marked predators immigrated to strawberry, rather than trap crops, resulting in a marked predator-to-Lygus spp. ratio of 5:1. Trap cropping effectively reduced the colonization of Lygus adults in strawberry. Converting weedy areas to native perennial plantings could further mitigate the risk of pest migration, while simultaneously conserving beneficial insects.

Behavioral Effects and Retention of Protein Immunomarkers on Plum Curculio Conotrachelus nenuphar (Coleoptera: Curculionidae).

Protein immunomarking can be used to track the dispersal of insects in the field or identify plant-insect interactions. By marking insects with known proteins and recapturing them, their movement or host use can be quantified with Enzyme Linked Immunosorbent Assay (ELISA). Before using this technique, retention and behavioral effects of these markers should be evaluated to ensure that the insect's natural behaviors are conserved. Here, we tested the effects of protein markers on the plum curculio, Conotrachelus nenuphar (Coleoptera: Curculionidae) using two different application methods. This weevil is native to North American and a pest of tree fruit and blueberry in the United States and causes damage resulting in near complete crop loss if left untreated. We tested the effects of marking adult C. nenuphar with two inexpensive food-based immunoprotein markers, bovine casein (cow's milk) and chicken albumin (egg whites) on climbing distance (total cm), lateral movement (total cm), and lateral movement speed (cm/s), as well as retention time of protein immunomarkers. Neither protein immunomarker affected C. nenuphar movement or climbing, although females climbed significantly greater distances than males. ELISA assays detected 37.5-56.2% of milk protein and 56.2-59.3% of egg on the insect 7 d after application depending on application method. Our findings indicate that food-based protein immunomarkers can be used in future studies to test C. nenuphar movement within host plants without impacting behavior. The use of protein immunomarking will allow studies that will lead to behaviorally based management tactics.

Arthropod Demography, Distribution, and Dispersion in a Novel Trap-Cropped Cotton Agroecosystem.

Vernonia [Vernonia galamensis (Cass.) Less.] (Asterales: Asteraceae) was examined as a potential trap crop for the cotton (Gossypium hirsutum L., Malvales: Malvaceae) arthropod complex. Four rows of vernonia were embedded within a 96-row cotton field. The abundance of true bug pests, true bug predators, and spiders were determined by whole-plant and sweep net sampling procedures during the early, middle, and late phases of the cotton-growing season. The census data showed that the arthropods had a strong preference for the vernonia trap crop throughout the cotton-growing season. The movement of the arthropods from the trap crop into cotton was also measured using the protein immunomarking technique as a mark-capture procedure. The arthropods inhabiting the vernonia trap crop were marked directly in the field with a broadcast spray application of egg albumin (protein) during each phase of the study. In turn, the captured specimens were examined for the presence of the mark by an egg albumin-specific enzyme-linked immunosorbent assay. Very few marked specimens were captured beyond the vernonia trap crop 1, 3, and 6 d after each marking event. The arthropods' strong attraction and fidelity to vernonia indicate that it could serve as a trap crop for cotton pests and a refuge for natural enemies.

Stable Isotope Marking of Laboratory-Reared Aedes aegypti (Diptera: Culicidae).

The use of stable isotope enrichment to mark mosquitoes has provided a tool to study the biology of vector species. In this study, we evaluated isotopic marking of Aedes aegypti (L.) (Diptera: Culicidae) in a laboratory setting. We determined the optimal dosage for marking adult Ae. aegypti mosquitoes with 13C and 15N. Additionally, Ae. aegypti mosquitoes were single and dually marked with 13C and 15N for up to 60 d postemergence without changes to adult body size or transgenerational marking. This report adds to the growing literature that explores the use of alternative marking methods for ecological and vector biology studies.

Laboratory Evaluation of Egg White and Milk External Biomarkers for Wasmannia auropunctata (Hymenoptera: Formicidae).

Acquisition and retention of two protein markers were tested on little fire ants, Wasmannia auropunctata Roger. Pure (100%) cow's milk and a dilution (10%) of chicken egg whites were applied to W. auropunctata directly by contact spray plus residue or indirectly via residual contact only with protein-marked plant debris. Protein-marked ants were held in plastic shoe-box-sized containers, collected at 0, 24, and 48 h after exposure to their respective marks, and then examined for the presence of the marks by a chicken egg albumin and milk casein-specific enzyme-linked immunosorbent assay. Cross-contamination rates were assessed by allowing ants marked with egg whites to interact with an equal number marked milk for 24 and 48 h, and then collected either individually or in bulk. Results indicated that the egg white biomarker was retained longer than milk and that more ants were successfully marked when the direct spray application method was employed. Cross-contamination rates were highest among bulk-collected ants and lowest among ants collected individually after 24 h. However, the rates of cross-contamination among individually collected ants increased and were similar to that of bulk-collected ants after 48 h. On the basis of our results, external protein marking may not be suitable if mass trapping is required or if the study extends beyond 24 h due to high cross-contamination rates among specimens collected in bulk and reduced marker detection rates.

Egg Albumin as a Protein Marker to Study Dispersal of Noctuidae in the Agroecosystem.

Knowledge of dispersal and spatial dynamics of pest populations is fundamental for implementation of integrated pest management and integrated resistance management. This study evaluated 1) the effectiveness of egg white albumin protein to mark larvae and adults of two polyphagous and highly mobile pests, Spodoptera frugiperda (J.E. Smith) (fall armyworm) and Helicoverpa zea (Boddie) (corn earworm) (Lepidoptera: Noctuidae), and 2) the sensitivity of polyvinylidene difluoride membrane (dot blot) in detecting albumin on marked insects. Laboratory and field experiments tested egg albumin as a protein marker, which was detected using two enzyme-linked immunosorbent assay (ELISA), microplate, and dot blot. In the laboratory, 100% of the moths sprayed with 20% egg white solution acquired the albumin marker, which was detected through the last time point tested (5 d) after application. Egg albumin was not effective at long-term marking of larvae, detected only prior the molting to the next instar. Albumin application in field cages resulted in a high percentage of moths detected as marked at 24 h and 5 d for both species. Egg albumin applied in the open field resulted in 15% of the recaptured corn earworm moths marked with most of them collected 150 m from the application area, although some were captured as far as 1,600 m within approximately 6 d after adult emergence. The results indicated egg albumin is a suitable marker to study the dispersion of fall armyworm and corn earworm in the agroecosystem and dot blot was as effective to detect egg albumin as was indirect ELISA.

Marking mosquitoes in their natural larval sites using 2H-enriched water: a promising approach for tracking over extended temporal and spatial scales.

BACKGROUND: 1.Tracking mosquitoes using current methods of mark-release-recapture are limited to small spatial and temporal scales exposing major gaps in understanding long-range movements and extended survival. Novel approaches to track mosquitoes may yield fresh insights into their biology which improves intervention activities to reduce disease transmission.Stable isotope enrichment of natural mosquito breeding sites allows large-scale marking of wild mosquitoes absent human handling. Mosquito larvae that develop in 2H-enriched water are expected to be detectable for over four months using tissue mass-fraction 2H measurements, providing opportunities for long-term mark-capture studies on a large scale. APPROACH: 2.A laboratory study followed by a field experiment of mosquito larval habitat 2H-enrichment was conducted in Mali, to evaluate potential labeling of wild mosquitoes. Twelve natural larval sites were enriched using [2H]-Deuterium-oxide (D2O, 99%). Enrichment level was maintained by supplementation following dilution by rains. Availability of 2H to mosquito larvae was enhanced by locally collected and cultured microorganisms (i.e. protozoa, algae and bacteria) reared in deuterated water, and provided as larval diet. Putative natural predators were removed from the larval sites and first instar larvae Anopheles gambiae s.l. larvae were added every other day. Emergence traps enabled collection of eclosing adults. Adult mosquitoes were kept at laboratory conditions for analysis of label attrition with age. RESULTS: 3.Deuterium enrichment of wild mosquitoes above background levels (maximum = 143.1 ppm) became apparent 5-6 days after initial exposure, after which 2H values increased steadily until ~24 days later (to a mean of approx. 220 ppm). Anopheles and Culex mosquitoes showed significantly different 2H values (211 and 194.2 ppm respectively). Both genera exhibited exponential label attrition (e (-x)) amounting to 21.6% by day 30 post emergence, after which attrition rate continuously decreased. Males of both taxa exhibited a higher mean 2H value compared to females. CONCLUSIONS: 4.Deuterium-oxide proved useful in marking mosquitoes in their natural larval sites and although costly, may prove valuable for studies of mosquitoes and other aquatic insects. Based on our field study, we provide a protocol for marking mosquito larval sites using deuterium-oxide.

Estimating the size of an open population using sparse capture-recapture data.

Sparse capture-recapture data from open populations are difficult to analyze using currently available frequentist statistical methods. However, in closed capture-recapture experiments, the Chao sparse estimator (Chao, 1989, Biometrics 45, 427-438) may be used to estimate population sizes when there are few recaptures. Here, we extend the Chao (1989) closed population size estimator to the open population setting by using linear regression and extrapolation techniques. We conduct a small simulation study and apply the models to several sparse capture-recapture data sets.

Intercrop movement of convergent lady beetle, Hippodamia convergens (Coleoptera: Coccinellidae), between adjacent cotton and alfalfa.

A 2-year study was conducted to characterize the intercrop movement of convergent lady beetle, Hippodamia convergens Guerin-Meneville (Coleoptera: Coccinellidae) between adjacent cotton and alfalfa. A dual protein-marking method was used to assess the intercrop movement of the lady beetles in each crop. In turns field collected lady beetles in each crop were assayed by protein specific ELISA to quantify the movement of beetles between the crops. Results indicated that a high percentage of convergent lady beetles caught in cotton (46% in 2008; 56% in 2009) and alfalfa (46% in 2008; 71% in 2009) contained a protein mark, thus indicating that convergent lady beetle movement was largely bidirectional between the adjacent crops. Although at a much lower proportion, lady beetles also showed unidirectional movement from cotton to alfalfa (5% in 2008 and 6% in 2009) and from alfalfa to cotton (9% in 2008 and 14% in 2009). The season-long bidirectional movement exhibited by the beetles was significantly higher in alfalfa than cotton during both years of the study. The total influx of lady beetles (bidirectional and unidirectional combined) was significantly higher in alfalfa compared with that in cotton for both years. While convergent lady beetles moved between adjacent cotton and alfalfa, they were more attracted to alfalfa when cotton was not flowering and/or when alfalfa offered more opportunities for prey. This study offers much needed information on intercrop movement of the convergent lady beetle that should facilitate integrated pest management decisions in cotton utilizing conservation biological control.