Manipulation of genes involved in regulating pigmentation can create externally visible markers of successful gene editing. However, such engineered mutations can have secondary impacts on key traits that are undesirable, especially if the mutants are expected to compete with wild-type (WT) individuals, such as might occur with a genetic sterile insect technique. Here, we tested for negative impacts in Lygus hesperus Knight from the knockout of aralkylamine N-acetyltransferase (aaNAT), needed to convert dopamine to N-acetyldopamine (NADA) sclerotin. Knockdown of this gene was previously shown to produce entirely black versions of this significant crop pest. Here, we compared knockout (KO) and WT strains across numerous behavioral and physiological traits. In most regards, the 2 strains were quite similar, with adults exhibiting equivalent locomotor activity, sperm production, mating activity, and susceptibility to heat, desiccation, and predation. However, some differences were observed with the aaNAT KO strain relative to the WT strain; eggs were more likely to hatch, nymphs had higher rates of survival to adulthood, adults lived longer, females produced fewer eggs, and males had a higher rate of mating success. Overall, the results suggest the aaNAT KO is a suitable marker that may facilitate rapid screening of gene edits and the development of population suppression systems.

1. Temperature impacts many aspects of species biology and ecology. A continuing struggle for studies in thermal ecology is accurate assessment of critical thermal maxima CT max. Identifying when loss of equilibrium (LOE) occurs has been criticized for being too subjective. This is particularly true of small organisms, such as insects, where the loss of coordination can be difficult to observe. As such, ecologists have often used lack of movement as a proxy for LOE. 2. Here, we designed, tested, and present a guide to recycling surplus gas chromatographs for use as a thermal chamber that allows accurate and high throughput assessment of CT max at relatively low cost. 3. We found the GC to be an adequate heating chamber for thermal experiments. Installation of a rotating rack that can hold glass observation vials allows for rapid identification of loss of equilibrium in subjects. We evaluated the CT max of a common generalist predator in the Arizona cotton agroecosystem, Collops vittatus . Additional tests of static heat exposure also revealed that this chamber can be used for assessing the impacts of heat stress on predatory behavior. 4. We hope to encourage other ecologists to use this guide to recycle surplus laboratory equipment for use in thermal ecology studies. We believe that our thermal insect carousel can be used for CT max, lethal temperature, and behavioral bioassays. ### Competing Interest Statement The authors have declared no competing interest.

Knowledge of insect dispersal and long-distance migratory flight capacity and patterns represent key factors needed for risk assessment of invasive pest species, insecticide resistance management, and more effective pest control. Having operative tools to both mark and track insect pest movement is therefore critical to achieving such goals. Here, we describe a new procedure for marking Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), one of the most economically important crop pests in the United States. Adult H. zea moths were effectively marked using the liquid fluorophore cartax green, a persistent UV-fluorescent pigment, both directly by topical application and indirectly by briefly submerging pupae in the marking solution prior to adult emergence. Regardless of the application method, the cartax mark was retained on the moths throughout their entire adult lifespan. No mortality differences were observed between cartax green-marked and water-marked (control) moths. Additionally, using rotary flight mills, we found no significant differences in several flight parameters, including total number of flights, flight speeds, flight distances, or flight durations between unmarked and cartax-marked moths. Under laboratory conditions, we did observe the lateral transfer of different colored fluorophores between moths, indicating that undesirable marking could potentially occur. Moreover, we found that not all fluorophores were equally retained on H. zea moths, with cartax green remaining intact on moths longer than did a corresponding magenta fluorophore. The results show that cartax green fluorophore could be a practical marker for H. zea and other holometabolous species targeted for large-scale mark-release-recapture research.

Understanding insect dispersal helps us predict the spread of insect pests and their natural enemies. Dispersal can be studied by marking, releasing, and recapturing insects, known as mark–release–recapture (MRR). MRR techniques should be convenient, economical, and persistent. Currently, there are limited options for marking small parasitoids that do not impact their fitness and dispersal ability. We evaluated commercially available fluorescent markers used in forensics. These fluorophores can easily be detected by ultraviolet (UV) light, requiring minimal costs and labor to process the marked specimens. This fluorophore marking technique was evaluated with the pest Drosophila suzukii and three parasitoids: Trissolcus japonicus, Pachycrepoideus vindemiae, Ganaspis brasiliensis (=G. kimorum). We evaluated the persistence of the marks on all the insects over time and examined the parasitoids for impacts on longevity, parasitism, locomotor activity, and flight take-off. The green fluorophore marker persisted for over 20 days on all four species. Marking generally did not consistently reduce the survival, parasitism rate, locomotor activity, or take-off of the parasitoids tested. Marked T. japonicus were recaptured in the field up to 100 m away from the release point and three weeks after release, indicating that this technique is a viable method for studying parasitoid dispersal.

The liquid fluorescent material SmartWater (R) has recently been reported as an effective external mark for arthropods. In this study, we examined the behavioral effects of the SmartWater marker on Lygus hesperus (Knight) (Hemiptera: Miridae). Specifically, we quantified the roaming and flight characteristics of unmarked, water-marked, and SmartWatermarked specimens. The results showed no significant differences in L. hesperus roaming and flight speeds, distances, and durations between the marking treatments. The results reported here and from previous research show the SmartWater product has enormous potential for mark-release-recapture type research.

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.

Mark-release-recapture studies require the application of a taggant to the arthropod under investigation before release at the study site so that recaptured specimens are identifiable. This mark must not affect the dispersal or foraging behaviour of the targeted organism. In this study, groups of Eretmocerus eremicus Rose and Zolnerowich (Hymenoptera: Aleyrodidae), a whitefly parasitoid, were marked externally with fluorescent dust or a liquid protein, or internally with a protein incorporated in their diet. Subsequently, the mark treatments' effects on foraging behaviour were compared with unmarked parasitoids. For the most part, marked specimens behaved similarly to their unmarked counterparts. However, there was an increase in host feeding, probing, and grooming activities exhibited by parasitoids exposed to certain mark treatments.

Flight mills are widely used to investigate insect flight behavior. As technology advances, the means to build a computerized control system for a flight mill has become more accessible in terms of both price and availability of components. However, the specialized electronics and programming knowledge required to build such a system can still present an obstacle to interested parties. Here, we describe a simple and inexpensive flight mill control system that can be easily assembled and operated without specialized experience. The hardware and software components are built around an Arduino single-board microcontroller, which outputs raw data in the form of timestamped detections of rotations of the flight mill arm. This control system is suitable both as the basis for new flight mills and for replacing outdated computer controls on existing flight mills. Additionally, it can be used with any rotary flight mill design that uses an electronic sensor to count rotations.

A marking and recapture sampling method was developed that shows promise for studying the dispersal behavior of small and delicate arthropods. Adult sweetpotato whiteflies, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), were externally marked with a liquid fluorophore that glows brightly under ultraviolet (UV) light. Then, a series of simulated recapture tests on fluorophore-marked whiteflies using yellow sticky cards were conducted. The marked whiteflies captured on the sticky cards were detected by direct visual inspection of photographs taken of the cards under white light and UV light. Whitefly counts taken under white and UV light were almost identical, implying high marking efficacy and visual distinctiveness. These results suggest that this fluorophore marking and sampling method could eliminate the tedious task of removing specimens from sticky cards and examining them individually for the presence of a mark.

We evaluated a method for marking arthropods that could serve as a valuable tool for mark-release-recapture dispersal research. The taggants tested consisted of three liquid fluorophores labeled cartax green, magenta, and orange. The manufacturer markets these fluorescent markers as forensic theft deterrents. Specimens of 16 genera of arthropods were externally marked with either one of the colored fluorophores or with water (negative control treatment). The specimens were then qualitatively and quantitatively inspected for fluorescence 24 h later. For the qualitative analysis, three independent observers scored each specimen by direct observation for the presence of a fluorescent mark. The specimens were scored using a portable ultraviolet (UV) tube lantern and a specialized NIGHTSEA-brand LED UV light. The three fluorophores were readily detected on many but not all the species examined, regardless of the type of UV light used. Moreover, the NIGHTSEA LED light yielded fewer false-negative observer errors than the lantern. Each specimen's fluorescence was measured with an automated dual-wavelength microplate fluorometer for the quantitative analysis. Overall, the quantitative analysis was very reliable at detecting fluorescence on a few taxa [e.g., Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), Chrysoperla spp. (Neuroptera: Chrysopidae)], effective on most taxa, and unreliable on several others [e.g., Collops vittatus (Say) (Coleoptera: Melyridae), Geocoris spp. (Hemiptera: Geocoridae), Mecaphesa celer (Hentz) (Araneae: Thomisidae)]. The cartax green marker was more readily detected than the magenta and orange markers with both visual and automated detection. Overall, the results show that these fluorophores could be effective markers for many arthropod species.

Conservation biological control is a fundamental tactic in integrated pest management (IPM). Greater biological control services can be achieved by enhancing agroecosystems to be more favorable to the presence, survival, and growth of natural enemy populations. One approach that has been tested in numerous agricultural systems is the deployment of synthetic chemicals that mimic those produced by the plant when under attack by pests. These signals may attract arthropod natural enemies to crop habitats and thus potentially improve biological control activity locally. A 2-yr field study was conducted in the cotton agroecosystem to evaluate the potential of synthetic methyl salicylate (MeSA) to attract native arthropod natural enemies and to enhance biological control services on two key pests. Slow-release packets of MeSA were deployed in replicated cotton plots season long. The abundance of multiple taxa of natural enemies and two major pests were monitored weekly by several sampling methods. The deployment of MeSA failed to increase natural enemy abundance and pest densities did not decline. Predator to prey ratios, used as a proxy to estimate biological control function, also largely failed to increase with MeSA deployment. One exception was a season-long increase in the ratio of Orius tristicolor (White) (Hemiptera: Anthocoridae) to Bemisia argentifolii Bellows and Perring (= Bemisia tabaci MEAM1) (Hemiptera: Aleyrodidae) adults within the context of biological control informed action thresholds. Overall results suggest that MeSA would not likely enhance conservation biological control by the natural enemy community typical of U.S. western cotton production systems.

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.

We examined the feasibility of externally marking insects with the liquid fluorescent forensic theft deterrent, SmartWater (SmartWater CSI, LLC.). We sprayed captive Lygus hesperus (Knight) (Hemiptera: Miridae), Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), and Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae) with SmartWater fluorophore, and then qualitatively examined them for fluorescence by visual inspection under ultraviolet (UV) light and quantitatively measured them with a multiwavelength microplate fluorometer. The results indicate that this product has enormous potential as a taggant for L. hesperus and B. tabaci. However, the marking efficiency for H. convergens was only adequate. The advantages and limitations of using SmartWater as a biological marker for arthropod mark-release-recapture research are discussed.

A molecular gut analysis technique is described to identify predators of Lygus hesperus (Knight), a significant pest of many crops. The technique is unique because it can pinpoint which life stage of the pest was consumed. Sentinel egg masses designed to mimic the endophytic egg-laying behavior of L. hesperus were marked with rabbit serum, while third instar and adult L. hesperus were marked with chicken and rat sera, respectively. Then, the variously labeled L. hesperus life stages were introduced into field cages that enclosed the native arthropod population inhabiting an individual cotton plant. After a 6-h exposure period, the predator assemblage, including the introduced and native L. hesperus population, in each cage were counted and had their gut contents examined for the presence of the variously marked L hesperus life stages by a suite of serum-specific enzyme-linked immunosorbent assays (ELISA). The whole-plant sampling scheme revealed that Geocoris punticpes (Say) and Geocoris pallens Stal (Hemiptera: Geocoridae) and members of the spider complex were the numerically dominant predator taxa in the cotton field.The gut content analyses also showed that these two taxa appeared to be the most prolific predators of the L. hesperus nymph stage. Other key findings include that Collops vittatus (Say) (Coleoptera: Melyridae) and Solenopsis xyloni McCook (Hymenoptera: Formicidae) appear to be adept at finding and feeding on the cryptic L. hesperus egg stage, and that L. hesperus, albeit at low frequencies, engaged in cannibalism. The methods described here could be adapted for studying life stage-specific feeding preferences for a wide variety of arthropod taxa.

Understanding the dispersal ability of invasive insects provides useful insights for developing effective management strategies. Historically, methods for marking insects for dispersal studies have been expensive, time-consuming, labor-intensive, and oftentimes ineffective, especially for woodboring beetles. Also, capturing or rearing insects requires human handling, which can alter behavior. Protein immunomarking is a well-established technique for studying the dispersal of insects; however, it has not been applied to woodborers. This study evaluates the potential for using protein immunomarkers applied directly to woodborer-infested trees to mark emerging beetles. Specifically, in the first experiment, we sprayed varying concentrations of ovalbumin (egg white) solution directly onto logs infested with emerald ash borer (EAB),Agrilus planipennisFairmaire (Coleoptera: Buprestidae, Agrilini). In turn, an enzyme-linked immunosorbent assay was used to detect the presence of protein on emerged beetles. To test the persistence of the mark, we applied varying concentrations of albumin to freeze-killed beetles, mounted them on pins, and placed them over various time intervals in an exposed location outdoors. Adult EAB self-marked as they emerged from protein-treated trees, with higher protein concentrations persisting for longer on the cuticle. This technique offers a convenient, inexpensive, and durable means of marking woodborers and circumvents the need for human handling, allowing for more natural behavior and more realistic estimates of dispersal. Protein self-marking may find application in studies of woodborer dispersal within natural forest environments.

A follow-up study was conducted to further evaluate the marking efficiency of broadcast spray applications of egg albumin (from chicken egg whites) on Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae) in alfalfa. A previous study recorded exceptional marking efficiency (e.g., >95% of the population) on H. convergens when using relatively high concentrations (10 to 50%) of chicken egg whites. The present study examines marking efficiency of egg whites using lower concentrations of 2.5, 5.0, and 10.0%. We used cadaver and free-roaming beetles to measure protein mark acquisition (and retention) of each protein concentration by direct contact with the spray application and incidental contact with protein residue on the plant tissue, respectively. The vertical distribution of the protein mark was also determined by sampling the upper and lower portions of the alfalfa canopy. The data indicate, regardless of the egg white treatment, that the backpack sprayer provided uniform coverage of egg albumin on the alfalfa plants and cadaver beetles. Also, almost every free-roaming beetle acquired a mark within 24 h after contact exposure to protein marked plants. This study shows that a very low concentration of egg albumin is sufficient for marking arthropods directly in the field.

A predator gut analysis technique is described that can simultaneously pinpoint predation events which are life stage-specific, intraspecies-specific (cannibalism) and interspecies-specific (intraguild). The third and fifth larval life stages of green lacewing, Chrysoperla carnea Stephens s.l. (Neuroptera: Chrysopidae), were marked with rabbit IgG and chicken IgY, respectively. The uniquely marked lacewing life stages were then introduced into caged arenas ( n = 59 caged experimental units) containing a cotton plant and an assemblage of generalist predators. The predators released into each arena were recaptured after 6 h and their gut contents were examined for the presence of rabbit IgG- and chicken IgY-marked lacewing remnants by an anti-rabbit and anti-chicken enzyme-linked immunosorbent assay (ELISA), respectively. The predator gut ELISAs detected one cannibalism event and 14 and 8 intraguild predation (IGP) events on third and fifth instar lacewings, respectively. This proof-of-concept study shows that this universal prey immunomarking technique (UFIT), when combined with field cage methods, can be useful for pinpointing cannibalism and life stage-specific predation events.

Alfalfa, Medicago sativa L. (Fabaceae), is a highly preferred host plant of Lygus spp. (Hemiptera: Miridae). As such, intercropping alfalfa trap-crops in strawberry production can serve as a sink for both Lygus (primarily Lygus hesperus Knight) and its natural enemies. Here we investigated the population dynamics and dispersal characteristics of the generalist predator complex in strawberry fields with alfalfa trap-crops spaced 50 rows (62 m) apart. Predator abundance was determined by counting six focal taxa collected from strawberry and alfalfa. The data revealed that Orius spp. (Hemiptera: Anthocoridae) were the numerically dominant predator taxa, comprising 84% of the focal predator population. In general, the population densities obtained for the various taxa throughout this agroecosystem were unexpectedly uniform. Predator movement from a central alfalfa trap-crop row was determined using a protein mark-capture procedure. Most protein-marked predator specimens were collected less than 2 m from the centrally marked alfalfa row, indicating that the trap-crop often produces a predator sink. Results suggest that alfalfa is a useful cultural (trap-cropping) and a biological (refuge for natural enemies) control tactic for managing Lygus spp. in strawberries.

Having an effective method to track movement of arthropods in nature is essential for any mark-release-recapture (MRR) or mark-capture (MC) type experiment. A simple protein immunomarking technique (PIT) was described over a quarter of a century ago that has since been proven to be a highly useful and versatile tool for tracking arthropod dispersal patterns. The PIT consists of tagging arthropods with a specific protein. In turn, recaptured arthropods are examined for the presence of the protein tag by a highly sensitive and specific enzyme-linked immunosorbent assay. In this article, I review the progression of the PIT procedure, provide guidelines for conducting a successful PIT (MRR or MC) dispersal study, and highlight some of the ways this procedure has been adapted to study the dispersal patterns of a wide variety of arthropod species. My goal is that this information will provide researchers with the motivation to develop even more creative uses for the PIT.

A universal food immunomarking technique (UFIT) is described for postmortem gut analysis detection of predation on the egg stage of Lygus hesperus Knight (Hemiptera: Miridae). Collops vittatus Say (Coleoptera: Melyridae) and Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae) were fed a single L. hesperus egg that was marked with rabbit and chicken sera proteins. The protein-marked egg remnants were detectable in the guts of the majority of the predators by each sera-specific enzyme-linked immunosorbent assay (ELISA) for 3 to 6 h after a feeding event. A novel technique was then developed to expose protein-marked eggs to predators that simulated the L. hesperus endophytic oviposition behavior. The procedure entailed embedding L. hesperus eggs in an artificial substrate that mimicked the stem of a plant. A predator feeding choice study was then conducted in cages that contained a cotton plant and artificial stems containing endophytic (concealed) and exophytic (exposed) egg patches. The endophytic and exophytic egg treatments were marked with chicken and rabbit protein, respectively. The gut analyses revealed that higher proportions of both predator populations contained remnants of the exophytic egg treatment and L. hesperus eggs were more vulnerable to C. vittatus than H. convergens. This study shows how the UFIT can be used to pinpoint stage-specific feeding activity on two distinct egg exposure treatments (endophytic and exophytic) of the same species.