Infection, choice behavior, and cross infectivity of the sculpted damsel bug, Nabis roseipennis offered tarnished plant bug, Lygus lineolaris infected with entomopathogenic nematodes

Infection, choice behavior, and cross infectivity of the sculpted damsel bug, Nabis roseipennis offered tarnished plant bug, Lygus lineolaris infected with entomopathogenic nematodes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Infection, choice behavior, and cross infectivity of the sculpted damsel bug, Nabis roseipennis offered tarnished plant bug, Lygus lineolaris infected with entomopathogenic nematodes James P. Glover, Nathan Spaulding, Marissa I. Nufer, Justin George, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4529008/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The tarnished plant bug, Lygus lineolaris Palisot de Beauvois (Hemiptera: Miridae), is an economically important pest of row crops worldwide. Ten isolates of entomopathogenic nematodes (EPNs) (Rhabditida: Steinernematidae and Heterorhabditidae) were evaluated against the third instar nymphal stage of tarnished plant bug and its generalist predator, the sculpted damsel bug, Nabis roseipennis Reuter (Hemiptera: Nabidae) one of the most abundant and commonly encountered damsel bugs in cotton and soybean agroecoscapes across the Southeastern United States. The objectives of these experiments were to assess the infectivity of entomopathogenic nematodes (EPN) by direct topical exposure against the sculpted damsel bug and tarnished plant bug, and whether the predator prey-choice is affected by EPN infection, and if feeding on EPN infected tarnished plant bug (TPB) prey items could result in cross-infection of the predator. Mortality rates at a concentration of 200 infective juveniles (IJs)/ml significantly differed among isolates and insect species, ranging from 30–93% for tarnished plant bugs and sculpted damsels 6–38%, respectively. The third instars of L. lineolaris were more susceptible to the ten nematode isolates than N. roseipennis . Higher pathogenicity on the tarnished plant bug and low mortality potential make strains HbHP88, HbVS, Sc17c + e, and SfSN the most promising candidate for the biological control of L. lineolaris under lab and greenhouse conditions while preserving beneficial predators of the Southeastern United States. tri-trophic interactions predation entomopathogenic nematodes beneficial insects crop pests Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key Message Trophic impacts of EPN's on how predation is affected by infected prey has not been studied in detail Tarnished plant bugs were susceptible to all 10 EPN strains isolates within 72 hours Sculpted damsel bugs fed on infected prey significantly shorter times than healthy prey items Generalist predator never became infected with EPN’s despite feeding duration Tarnished Plant bug infected with nematodes are less attractive to predatory bugs aiding in biological control Introduction The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), is a major pest of row crops in the southern United States (Williams, 2015). Highly polyphagous, it feeds on a wide range of host plants, including the most economically important crops grown in the Midsouth (George et al. 2021 , Parys 2014 ). The tarnished plant bug has been recognized as a major economic pest of cotton, causing significant fruit loss and economic damage in cotton-producing regions across the mid-South, the Mississippi Delta region, and other areas of the world (Snodgrass et al. 2009 ). Pest status is exacerbated by a multivoltine life cycle. The sculpted damsel bug, Nabis roseipennis Reuter (Hemiptera: Nabidae), is among the most abundant and most common of the damsel bugs encountered in cotton and soybean across the Southeastern United States (Pfannenstiel et al. 1998; Irwin et al. 1980; Shepard et al. 1974 ; Pitre et al. 1978; and Dinkins et al. 1970 ). A complex of Nabids spp . are commonly found in traditional row crop agroecosystems, including cotton, soybean, and sorghum, and are predators of many Lepidopteran eggs, aphids, and other small soft-bodied insects (Coscarón et al. 2015 ; Stewart et al. 2007 ). Nymphal stages of many Nabid spp. can typically consume one or more eggs and or aphids per day, while more mature instars and adults may consume as many as two dozen prey items (Conti et al. 2021 ; Haseeb et al. 2018 ; and Bueno et al. 2012). Many species of Nabids irrespective of nymphal growth stage have been reported to feed on all life stages of Lygus spp. (Perkins and Watson 1972 ; Dumont et al. 2023 ; Perkins et al. 1971). Entomopathogenic nematodes (EPN's) are a group of parasitic nematodes that have been extensively studied for their potential use in the biological control of insect pests as well as a model for studying reproductive biology (Mahmoud, 2017). EPNs are obligate pathogens of insects and important biological control agents of insect pests (Pilz et al. 2014 ). EPN's in the genera Steinernema and Heterorhabditis , have shown great potential for biological pest control in specific agricultural settings. These nematodes are capable of infecting and killing insect pests, making them an attractive alternative to chemical pesticides, which can have negative effects on the environment and human health (Kaya and Gaugler, 1993 ). The use of nematodes as biological control agents has been widely researched, and EPNs have emerged as promising candidates (Kaya and Stock, 1997). EPN's have been found to be highly effective in controlling a variety of insect pests, including some of the most destructive pests in agriculture, such as the corn rootworm Diabrotica spp. and black vine weevil Otiorhynchus sulcatus (Toepfer et al. 2008 ; Lewis et al. 2005). In addition, some species of EPN 's have been found to be highly host-specific, which makes them ideal for use as biological control agents since they are believed to cause less off-target effects on organisms (Gaugler and Kayla 1990; Abd-Elgawad, 2019 ). Many species of EPN's have been shown to be effective against a wide range of insect pests, including white grubs, root weevils, and cutworms (Kaya and Gaugler, 1993 ). They have also been used successfully against soil-dwelling pests such as corn rootworms and wireworms (Stock and Goodrich-Blair, 2012 ). The use of EPN's has been shown to reduce insect pest populations and increase crop yields in specific agricultural settings (Kaya and Gaugler, 1993 ). Recently, Steenman et al. (2023) found that the EPN S. carpocapsae was an effective biological control agent against three common mirid pest species within a greenhouse environment. Furthermore, the Potential of Nabis americoferus as a biological control agent of Lygus lineolaris in strawberry fields has recently been reported (Dumont et al. 2023 ). Furthermore, EPN's have several advantages over traditional chemical insecticides. Nematodes are highly host-specific to their target pests, having considerably less off-target or non-target effects on other organisms, leaving beneficial insects unharmed (Stock and Goodrich-Blair, 2012 ; Kaya and Gaugler, 1993 .). They also have a short persistence in the environment, as they are vulnerable to desiccation and predation, which reduces the risk of accumulation in the food chain (Kaya and Gaugler, 1993 ). In addition, they do not pose a risk of developing resistance in pest populations (Stock and Goodrich-Blair, 2012 ). Nematodes are less likely to develop resistance to chemical insecticides, which can become less effective over time as pests evolve resistance. Furthermore, nematodes are environmentally friendly and do not leave harmful residues in the soil or water. The objectives of these experiments were to assess the infectivity and efficacy of two groups of entomopathogenic nematodes (EPN), Steinernema and Heterorhabditis by direct topical exposure against the tarnished plant bug and the generalist cotton predator, the sculpted damsel bug. Determine if predator prey-choice is affected by EPN infection, and if feeding on EPN infected tarnished plant bug (TPB) prey items could result in a cross-infection of the predator. The goal of this research is to determine the extent to which EPNs could infect and cause mortality in hosts and identify any strain specific differences. To gain further insight into the practical application and potential of EPNs to control TPB in a production setting, we conducted additional greenhouse experiments to further analyze potential cross-infectivity and predation of an infected prey item by a generalist predator. Materials and Methods Insect source and rearing All studies were conducted at the USDA-ARS Southern Insect Management Research Unit, Stoneville, MS. Laboratory-reared TPB and nabid colonies were established from field collections of mixed-age late instar nymphs and adults collected from primarily large stands of seedling Johnsongrass ( Sorghum halepense ) and supplemented with collections from other broadleaf weeds common across the Mid-south including Palmer amaranth ( Amaranthus palmeri ). TPB colonies were reared on a semi-solid autoclavable diet routinely used for the mass rearing of Lygus lineolaris (Portilla et al. 2011). Nabids were reared individually to adulthood in an environmental chamber (Percival Scientific Inc, Perry, Iowa) at a temperature of 27 ± 0.5°C, 60 ± 5% RH, and a 14:10 (L:D) h photoperiod. Individuals were supplied with a 42 ml plastic cup containing a small piece of cotton saturated with a 2% sucrose solution as a water source and freshly hatched Helicoverpa zea (Boddie) neonates as a live food source. EPN cultivation and insect inoculation The twenty EPN isolates were reared in the last instar larvae of the greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae), which were obtained from Josh's Frogs (Owosso, Michigan) (Table 1). The ratio of IJs to a host was 10 IJs/host. 200 IJs in 2 ml of aqueous suspension were pipetted to a 100 x 15 mm Petri dish with a single layer of VWR filter paper and 20 hosts. The petri dishes were then covered with a black plastic bag to protect them from light and incubated at room temperature (~ 22°C). After the hosts died from EPN infection, the cadavers were moved to White traps (Kaya and Stock, 1997) that were also kept at room temperature and coved with a black plastic bag. The traps were checked every day for the emergence of IJs, which were collected and stored in tissue culture flasks in a 13°C refrigerator. The concentration of infective juvenile was counted using a phase hemacytometer (Hausser Scientific #1475, Waltham, Massachusetts) and viewed under a Leica S9 stereo microscope (Leica Microsystems, Wetzlar, Germany) and adjusted by adding deionized water to attain a final concentration of 200 IJs per/ml. Aqueous nematode solutions of 200 IJs per 1ml of deionized water were pipetted directly onto the pronotum and mesothoracic regions of both adult sculpted damsel bugs and one-day old 3rd instar nymph tarnished plant bugs, respectively. Individuals were observed under a light microscope by removing the plastic lid from the 42 ml plastic assay cup containing diet and were examined for externally visible infections with any stage of EPN. After the duration of the experiment (10d) cadavers were retained and placed in cultivation flasks to incubate and determine EPN infection status. Table 1 List /codex of entomopathogenic species of nematodes (EPN's) used in a series of experiments presented. EPN species EPN origin EPN strain Codex Heterorhabditis georgiana Georgia, USA Kesha Hg Heterorhabditis bacteriophora Utah, USA HP88 Hb-HP88 Heterorhabditis bacteriophora Georgia, USA HVS Hb-HVS Steinernema rarum Combined from isolates found in Louisiana, USA and Mississippi, USA 17 c + e Sra Steinernema feltiae France SN Sf Steinernema carpocapsae Arkansas, USA Cxrd Sc-Cxrd Steinernema riobrave Texas, USA 355 Sri-355 Steinernema carpocapsae Georgia, USA A11 Sc-A11 Steinernema riobrave Texas, USA 7–12 Sri-712 Heterorhabditis floridensis Georgia, USA K22 Hf Lab and greenhouse prey preference trials Predator preference assays were designed according to Avery et al. (2022), in brief, prey preference behavior of the SDB adults was studied using choice arenas made from sterile Petri dishes (150 x 15 mm: Fisher Scientific, Inc. Waltham, MA, USA) lined with a round VWR® Grade 410 qualitative filter paper, (VWR-Avantor, Radnor, PA, USA) and trimmed to fit within the circumference of the bottom petri dish securely leaving no gaps. Arenas were designed with a central release zone centered on the drawn line using the cap from a 50 ml falcon plastic centrifuge tube placed in the center of each arena. Third instar tarnished plant bugs were topically treated with aqueous nematode solutions of 200 IJs per 1ml of deionized water were pipetted directly onto the pronotum and mesothoracic regions, held on green beans sections (15mm) previously triple washed with 10% NaClO, in petri dishes (52 x 5 mm) lined with a round filter paper of similar size for 24h before being offered to SDB adults. One live 24h infected tarnished plant bug and one live uninfected nymph (water control) were placed opposite and perpendicular to the principal axis line. Tarnished plant bug nymphs were brushed with a natural hair paint brush (#0) and placed at equal distances from the drawn line (12.7 mm). Featherweight forceps were used to transfer SDB adults from rearing cups to under the 50ml falcon tube cap with a circular hole punched (20mm) in the center (Fig. 1). Plastic infesting caps were removed once the SDB adult exited the hole in the cap and was freely moving in the arena on the filter paper. Petri dish choice assays were performed in a walk-in environmental chamber with 16: 8 h (L:D) photoperiod, 28°C, and 15% RH with arenas placed directly under overhead luminescent light. Predator preference trials resulted in one of three possible outcomes: feeding on an infected TPB prey, feeding on an uninfected TPB nymph, or no choice. A "predation event" was defined as a prolonged stylet insertion and feeding event lasting ≥ 60 seconds. Preference trials commenced and lasted 20 min once the SDB exited from the hole in the cap and made contact with the filter paper. Trial replicates received freshly cleaned and air-dried Petri dishes and caps (75% alcohol) supplied with a new filter paper. The orientation of the infected and uninfected prey types was randomly assigned to exclude any directional bias. Greenhouse experiments were performed on individual non-Bt cotton plants (DLP1892, delta pine, ark, USA) in two-gallon size black nursery greenhouse pots in standard potting media. SDB were released onto greenhouse-grown cotton in the second week of blooming (approx. 12 nodes). Cotton terminals were isolated with insect enclosure bags constructed from organza (22 x 22 x 8 mm ~ 240 µm mesh, JoAnn’s Fabrics, Hudson, OH) that enclosed the plant terminals affixed with pipe cleaners (top five nodes) containing many squaring sites. The second week of bloom (≈ 55d) was chosen to reflect not only a physiologically vulnerable growth stage of cotton but also a location that's commonly associated with economic damage. We conducted 20 replicates for the strains with mortality > 70% (strains Hb-HVS, Sra, Sf, Sc-Cxrd) (Fig. 2.). Plants were infested by carefully opening the enclosure and paint brushing a 24h infected TPB 3rd instar nymphs, one per cage, onto a fully mature and opened terminal leaf and allowing 10 min to acclimate and explore the canopy. Featherweight forceps were used to transfer SDB adults from rearing cups to caged plant terminals, placed on an opposing mature leaf, and the cage securely fastened after the TPB acclimation period. Cages were vigorously inspected to ensure insects were not immobilized by the folds in the fabric and were completely confined to the desired nodes of growth. Cages were inspected every 24h for predated TPB as evidenced by a shriveled and imploded body indicative of feeding similar to individuals fed on during the petri dish assays. Many cages inspected were observed with SDB actively feeding on or actively roaming and carrying the TPB nymph. Data analysis All experimental measurements of mortality and infection were analyzed as a randomized complete block with a factorial arrangement in PROC GLM (Littell et al. 1991) (10 EPN strains examined x 3rd instar tarnished plant bug and adult sculpted damsel bugs exposed plus a water control). Each treatment combination was repeated three times. A Tukey's mean separation test (α = 0.05) was used to compare results. Individuals who made no prey choice or who never excited the cap were excluded from the analysis. Predator preferences and time data (min) between infected and uninfected prey were analyzed by using a X 2 test of independence to estimate the difference in probability of choosing infected versus control for each strain at each time point, allowing time to be treated as a continuous variable with non-linear time trend. Results Infection and mortality of tarnished plant bug nymphs Third instar tarnished plant bug mortality at seven days was significantly affected by topical exposure with the ten EPN strains ( F = 93.69; df = 10, 418; P = < 0.0001), with mortality ranging from 57–93%. The EPN strains with the highest mortality observed across the study were Sra and Sc-Cxrd with 94%, 92%, respectively (Fig. 2). No significant interactions were detected ( P > 0.05) across this study. A Tukey's post hoc means separations test ( P < 0.05) (α = 0.05) determined that strains Hb-HP88, Hb-HVS, SRA, SF, and Sc-Cxrd caused significant mortality at five and seven days post topical exposure, and significant mortality on day three for Sri-355 when compared to strains Sc-A11, Sc-A11, HF, and controls (Fig. 2). Infection and mortality of adult nabids Adult nabid mortality one-week post exposure to the ten EPN strains was significantly higher for strains Sc-Cxrd and Sc-A11 treatments, and the highest mortality observed ranging from 27% -38%, respectively ( F = 7.19; df = 10, 316; P = 0.05). A Tukey's post hoc means separations test (P < 0.05) (α = 0.05) determined that nabids exposed to strain Sc-Cxrd experienced significant mortality at all three sampling points and on day seven for strain Sc-A11 post exposure (Fig. 3). All strains caused mortality > 5% excluding strains Hb-HP88, Hb-HVS, SRA, and HF where no activity was detected. Predator preference In the arena choice assays where nabids had a choice of uninfected tarnished plant bug prey item versus infected with strain Hb-HVS, 59.4% of individuals made no choice (n = 69). 32.8% of nabids fed on the control tarnished plant bug (topical water exposure) (n = 38), and 7.8% (n = 9) fed on an infected prey item (Fig. 4). Exposed and infected tarnished plant bug prey were attacked significantly less by adult nabids compared to uninfected prey. On average nabids consumed infected prey items for 45 s ± 13 s (range 30–75 s) compared to 321 ± 17 s (range 60 − 900 s) on the uninfected prey x 2 (1, N = 116) = 11.84, p < 0.0006. Nabids that received tarnished plant bug prey items infected with strain Sra or a control insect made no choice 56% (n = 105), while 40.6% (n = 76) fed on the control tarnished plant bug, and 3.2% (n = 6) fed on infected prey item (Fig. 4). Exposed and infected tarnished plant bug prey were attacked significantly less x 2 (1, N = 187) = 13.55, p < 0.0002 by adult nabids compared to uninfected tarnished plant bugs. The average time for a nabid to choose and feed on the EPN infected tarnished plant bug prey was 30 s, compared to 452 ± 25 s (range 12–900 s) for the nabids that chose the uninfected prey. Nabids, when offered a choice of infected, tarnished plant bug prey infected with strain SF or control, 49% nabids made no choice (n = 92), 33% fed on the control tarnished plant bug (n = 62), and 17% (n = 33) fed on infected prey item (Fig. 4). Exposed and infected tarnished plant bug prey were attacked significantly less by adult nabids compared to uninfected prey. The average time for a nabid to choose and feed on the EPN infected tarnished plant bug prey was 153 s, compared to 565 ± 15 s (range 12–900 s) for the nabids that chose the uninfected prey x 2 (1, N = 187) = 14.72, p < 0.0001. Nabids that were given tarnished plant bug prey items infected with Sc-Cxrd on an uninfected prey item had 39% (n = 73) of individuals made no choice, 44% (n = 82) feed on the control tarnished plant bug (topical water exposure), and 17% (n = 32) fed on an infected prey item. Exposed and infected tarnished plant bug prey were attacked significantly less by adult nabids compared to uninfected prey. The average time for a nabid to choose and feed on the Sc-Cxrd EPN infected tarnished plant bug prey was 62 s, compared to 528 ± 37 s (range 25–900 s) for the nabids that chose the uninfected prey x 2 (1, N = 187) = 11.63, p < 0.0006. Longest feeding of nabids on EPN infected tarnished plant bugs was observed with strain SF and strain Sc-Cxrd for 204 s and 180 s, respectively (Fig. 4). Green house no choice assay and cross-infectivity Nabids individually infested on greenhouse-grown cotton plants consumed > 80% of infected prey items in caged no-choice assays across the four strains tested (Fig. 5). Of the nabids who made a choice to feed on infected tarnished plant bug prey (n = 81) none of the individuals regardless of the duration of the feeding event become infected with any of the four EPN strains tested in this experiment (Table 1). Discussion First discovered in the early 1920s, interest in EPNs increased in the 1950s with commercialization of the fist species in the 1980s. Given the safety profile to humans, the environment, and non-target species EPNs have been excluded from pesticide registration hurdles (Ehlers 2005 , Piedra-Buena et al. 2015 ). Research on the application, biology, and ecology of EPNs began in the early1990s and further expanded into basic research in the 2000s. The extensive body of literature resulting from this research has resulted in several seminal books (Gaugler and Kaya, 1990; Bedding et al., 1993 ; Gaugler, 2002 ; Grewal et al., 2005 ). The Nabidae mouthpart or rostrum is composed of four flexible and mobile segments that sets at the mesocoxae at resting position. The first two joints are thick and rectilinear and are the most flexible followed by the third and generally longest segment (Pericart 1987). The relationship between nematodes specifically Steinernema and Heterorhabditis and their bacterial symbionts Xenorhabdus spp. and Photorhabdus spp., respectively are highly specific (Shaprio-llan et al. 2023). These bacteria are the primary causative agent responsible for killing the host insect and providing nutrition for the nematodes (Lewis & Clarke 2012 ; Singh et al. 2022 ). Photorhabdus luminescens is a bacterial insect pathogen from the Enterobacteriaceae family of soil-based nematodes from the genera Heterorhabditis . This bacterium produces an array of toxins that assemble as a complex (approximately 25 nm in length and 1.5 nm in diameter) (Alouf et al. 2005 ). EPN associated Xenorhabdus spp. bacteria have been reported to range in size approximately 2.6 ± 0.4 µm 3 (Wang et al. 2007 ). The average size of the smallest and forth section of the sculpted damsel bug was approximately 0.6 mm and 0.021 length and diameter, respectively (Glover unpublished data). Given the size of the symbiotic bacteria and the relative mouthpart size of the damsel bug it is theoretically possible for the microbes to be acquired. However, acquisition and transmission of EPN bacterial symbionts from the Enterobacteriaceae family remains unknown. Given the longevity of adult Nabis spp., which has been reported to be almost 60 days (Rebolledo et al. 2005 ), there is a dearth of information concerning its interactions with EPN's. So far, the trophic impact of EPN's on how predation is affected by infected prey items has not been studied in detail. The experimental data presented in this study is the first to investigate EPN associated mortality and feeding behavior of the sculpted damsel bug offered EPN infected prey items. Our data shows that the tarnished plant bug was susceptible to all ten EPN strains tested in the present study with mortality rates of more than 60% reached after seven days. However, strains Hb-HP88, Hb-HVS, Sra, Sf, and Sc-Cxrd showed marked mortality > 50% three days post-exposure to infective juveniles. Similarly, Steenman et al. (2023) found that three common mirid pest species were similarly susceptible to the EPN family S. carpocapsae (ScAll and ScCxrd). Many of the infected tarnished plant bug nymphs began to change body coloration to reddish-brown as infection progressed and may be a reliable indicator of EPN progressive infection (Fig. 6). Mortality for the sculpted damsel bug was significantly affected by strains Sc-Cxrd and strain Sc-A11 with mortality ranging from 25–38% respectively. Conversely, strains Hb-Hp88, Hb-HVS, Sra, and Hf had no significant effect on mortality. Based on our data, we choose strains Hb-HVS, Sra, SF, and Sc-Cxrd due to the high associated mortality on the tarnished plant bug and the relatively low or no mortality associated with the sculpted damsel bug. This selection allowed for the testing of four strains from the two major genus of EPN currently under investigation in modern agriculture. Our study indicates that the sculpted damsel bug primarily avoided EPN infected prey items when given a choice to predate a healthy prey item, and greater avoidance was observed after an individual made contact first with an infected prey item. Damsel bugs fed on infected prey items (81) across the entirety of the experiment using four strains tested. However, we found that no damsel bug had been infected with any strain or life stage of EPN across the strains tested here after consumption, regardless of the feeding duration of an infected EPN tarnished plant bug prey item. The results of this study may also provide insight into the general effects of EPNs on predator-prey choice. In this study, our generalist predator, the sculpted damsel bug, actively fed on and probed infected prey items for significantly shorter times than uninfected or healthy prey when offered a choice. The sculpted damsel generally avoided the infected tarnished plant bug, particularly after feeding and probing contact with an infected individual in choice arenas. Of the 81 instances where the predator fed on the infected prey item, more than half of the feeding events lasted for < 60 seconds, and the predator actively avoided infected prey in choice of the healthy prey item for the remainder of the assay. Cadavers hosting the nematode/bacteria complex have been shown to be highly deterrent for detritivorous insects and was fist demonstrated with the EPN Photorhabdus luminescens (Heterorhabditidae) and workers of Linepithema humile (Hymenoptera: Formicidae) (Baur et al. 1998 ). Mertz et al. ( 2015 ) demonstrated that the predator Calosoma granulatum actively avoided feeding on infected larvae of Spodoptera frugiperda when given a choice of healthy versus infected prey types. The sculpted damsel bug generally avoided the infected prey items in the choice experiments presented here. This observation may translate to field avoidance of EPN infected prey. It may further facilitate an even greater number of predation events of uninfected prey, leading to increased predation of tarnished plant bugs in a field setting. However, greenhouse assays demonstrated that under no-choice conditions that predators would actively search and predate infected tarnished plant bugs > 75% with some avoidance observed, however no predators ever become externally or internally infected with any stage of EPN strain tested. The results obtained in the present experiments show that in general, the of the ten EPN isolates Hb-HVS, Sra, SF, and Sc-Cxrd have high potential for use in biological control against the TPB, are safe for the native generalist predator N. roseipennis. Further investigation in a field setting will provide additional behavioral information on how potential volatile bouquets influence prey visitation and predation events (Zhang et al. 2019 ). In conclusion, the use of EPN's as a biological control method in agriculture shows great promise. They are effective against a wide range of insect pests, specific to their target pests, and have several advantages over chemical pesticides. As such, further research on the development of encapsulation and U.V. protectant technologies will increase the efficiency of biological control of pests in an integrated pest management (IPM) system. Declarations Funding: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Author Contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by James P. Glover, Justin George and Gadi V.P. Reddy. The first draft of the manuscript was written by James Paul Glover and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability Statement: Not applicable. Ethics approval: Research involving human participants and/or animals, Not applicable. Consent to participate: Informed consent was obtained from all individual participants included in the study. Consent to publish: Not applicable. References Abd-Elgawad MM (2019) Towards optimization of entomopathogenic nematodes for more service in the biological control of insect pests. Egypt J Biol Pest Control 29:1–8 Alouf, J. E., Ladant, D., & Popoff, M. R. (2005) The comprehensive sourcebook of bacterial protein toxins . Elsevier Baur ME, Kaya HK, Strong DR (1998) Foraging ants as scavengers on entomopathogenic nematode-killed insects. Biol Control 12:231–236 Bedding, R. A., Akhurst, R. J., & Kaya, H. K. (1993) Nematodes and the biological control of insect pests . Csiro Publishing. pp 1350-1356. Braman SK, Yeargan KV (1990) Phenology and abundance of Nabis americoferus , N. roseipennis , and N. rufusculus (Hemiptera: Nabidae) and their parasitoids in alfalfa and soybean. J Econ Entomol 83:823–830 Bueno VHP, van Lenteren JC (2012) Predatory bugs (Heteroptera), in Insect Bioecology and Nutrition for Integrated Pest Management , ed. by AR Panizzi and J Parra. CRC Press, Boca Raton, FL, pp. 539–569 Burnell, A., & Stock, S. P. (2000) Heterorhabditis, Steinernema and their bacterial symbionts—lethal pathogens of insects. Nematology , 2 : 31-42 Campbell JF, Gaugler R, (1993) Entomopathogenic nematodes: a review of biology, behavior and host-parasite interactions. J Nematol 25:235–249 Campos–Herrera, R., Barbercheck, M., Hoy, C. W., & Stock, S. P. (2012) Entomopathogenic nematodes as a model system for advancing the frontiers of ecology. Journal of nematology , 44 :162 Catchot A, Musser F, Gore J, Cook D, Daves C, Lorenz G, Studebaker G (2009) Midsouth Multistate Evaluation. Conti E, Avila G, Barratt B, Cingolani F, Colazza S, Guarino S, Wajnberg E (2021) Biological control of invasive stink bugs: Review of global state and future prospects. Entomol Exp Appl 169:28–51 Coscarón MC, Braman SK, Cornelis M (2015) Damsel Bugs (Nabidae). In: Panizzi, A., Grazia, J. (eds) True Bugs (Heteroptera) of the Neotropics. Entomology in Focus, vol 2. Springer, Dordrecht. https://doi.org/10.1007/978-94-017-9861-7_11 Daoust, SP, King KC, Brodeur J, Roitberg BD, Roche B, Thomas F (2015) Making the best of a bad situation: host partial resistance and bypass of behavioral manipulation by parasites? Trends Parasitol 31:413–418 Dinkins RL, Brazzel JR, Wilson CA (1970) Seasonal incidence of major predaceous arthropods in Mississippi cotton fields. J Econ Entomol 63:814–817 Dumont, F., Solà, M., Provost, C., & Lucas, E. (2023) The potential of Nabis americoferus and Orius insidiosus as biological control agents of Lygus lineolaris in strawberry fields. Insects , 14 (4), 385. Ehlers, R. U. (2005) Forum on safety and regulation: Nematodes as biocontrol agents. Wallingford UK: CABI publishing, 107-114 Gaugler, R. (1990) Entomopathogenic nematodes in biological control (Vol. 227). H. K. Kaya (Ed.). Boca Raton: CRC press Gaugler, R. (2002) Entomopathogenic Nematology. Wallingford, UK: CABI Publishing. doi: 10.1079/9780851995670.0000 George J, Glover JP, Gore J, Crow WD, Reddy GV (2021) Biology, ecology, and pest management of the tarnished plant bug, Lygus lineola ris (Palisot de Beauvois) in southern row crops. Insects 12:807 Godfrey KE, Whitcomb WH, Stimac JL (1989) Arthropod predators of velvetbean caterpillar, Anticarsia gemmatalis HÃubner (Lepidoptera: Noctuidae), eggs and larvae. Environ Entomol 18:118–123 Grewal, P. S., Ehlers, R. U., & Shapiro-Ilan, D. I. (Eds.). (2005) Nematodes as biocontrol agents . CABI Haseeb M, Gordon TL, Kanga LH, Legaspi JC (2018) Abundance of natural enemies of Nezara viridula (Hemiptera: Pentatomidae) on three cultivars of sweet alyssum. J Appl Entomol 142:847–853 Irwin ME, Shepard M (1980) Sampling Predaceous Hemiptera on Soybean. In: Kogan, M., Herzog, D.C. (eds) Sampling Methods in Soybean Entomology. Springer Series in Experimental Entomology. Springer, New York, NY. https://doi.org/10.1007/978-1-4612-9998-1_25 Kaya HK, Gaugler R (1993) Entomopathogenic nematodes. Ann Rev Entomol 38:181–206. Lewis, E. E., & Clarke, D. J. (2012) Nematode parasites and entomopathogens. In Insect pathology (pp. 395-424). Academic Press Lewis, E. E., & Grewal, P. S. (2005) Interactions with plant-parasitic nematodes Mertz, N. R., Agudelo, E. J. G., Sales, F. S., & Moino Junior, A. (2015) Effects of entomopathogenic nematodes on the predator Calosoma granulatum in the laboratory. Journal of insect behavior , 28 , 312-327. Parys KA (2014) Host plants of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois). Beltwide Cotton Conferences, 6–8 January 2014, National Cotton Council Perkins, P. V., & Watson, T. F. (1972) Biology of Nabis alternatus (Hemiptera: Nabidae). Annals of the Entomological Society of America , 65 (1), 54-57. Péricart, J. (1987) Hémiptères Nabidae d'Europe occidentale et du Maghreb . Fédération française des sociétés de sciences naturelles Pilz C, Toepfer S, Knuth P, Strimitzer T, Heimbach U, Grabenweger G (2014) Persistence of the entomoparasitic nematode Heterorhabditis bacteriophora in maize fields. J Appl Entomol 138:202–212 Piedra-Buena, A., López-Cepero, J., & Campos-Herrera, R (2015) Entomopathogenic nematode production and application: regulation, ecological impact and non–target effects. In Nematode Pathogenesis of Insects and Other Pests: Ecology and Applied Technologies for Sustainable Plant and Crop Protection (pp. 255-282). Cham: Springer International Publishing Pfannenstiel RS, Yeargan KV (1998) Ovipositional preference and distribution of eggs in selected field and vegetable crops by Nabis roseipennis (Hemiptera: Nabidae). J Entomol Sci 33:82–89 Portilla, M., G. Snodgrass, and D. Streett (2010) Effect of modification of the NI artificial diet on the biological fitness parameters of mass reared western tarnished plant bug, Lygus hesperus. J. Insect Sci. 11: 1–10. Rebolledo R, Villegas G, Klein C, Aguilera A (2005) Fluctuación poblacional, capacidad depredadora y longevidad de Nabis punctipennis Blanchard (Hemiptera: Nabidae). Agric Técnica , 65:442–446 Shapiro-Ilan, D. I., Leite, L. G., & Han, R. (2023) Production of entomopathogenic nematodes. In Mass production of beneficial organisms (pp. 293-315). Academic Press Shapiro-Ilan, D., & Dolinski, C. (2015) Entomopathogenic nematode application technology. Nematode Pathogenesis of Insects and Other Pests: Ecology and Applied Technologies for Sustainable Plant and Crop Protection , 231-254 Shapiro-Ilan DI, Gouge DH, Piggott SJ, Fife JP (2006) Application technology and environmental considerations for use of entomopathogenic nematodes in biological control. Biol Control 38:124–133 Shapiro-Ilan, D. I., Gouge, D. H., Koppenhöfer, A. M., & Gaugler, R. (2002) Entomopathogenic nematology. Wallingford: CABI Publishing , 333-355 Shepard M, Carner GR, Turnipseed SG (1974) Seasonal abundance of predaceous arthropods in soybeans. Environ Entomol 3:985–988 Singh, A. K., Kumar, M., Ahuja, A., Vinay, B. K., Kommu, K. K., Thakur, S., ... & Parihar, M. (2022) Entomopathogenic nematodes: A sustainable option for insect pest management. In Biopesticides (pp. 73-92). Woodhead Publishing Snodgrass G, Gore J, Abel C, Jackson R (2009) Acephate resistance in populations of the tarnished plant bug (Heteroptera: Miridae) from the Mississippi River Delta. J Econ Entomol 102:699–707 Stewart SD, Layton B, Catchot A (2007) Common beneficial arthropods found in field crops. University of Tennessee Extension. http://msucares. com/pubs/publications/e0020. Pdf Stock SP, Goodrich-Blair H (2012) Entomopathogenic nematodes and their bacterial symbionts: the inside out of a mutualistic association. Symbiosis 58:47–65 Toepfer, S., Peters, A., Ehlers, R. U., & Kuhlmann, U. (2008) Comparative assessment of the efficacy of entomopathogenic nematode species at reducing western corn rootworm larvae and root damage in maize. Journal of Applied Entomology , 132:337-348 Wang, Y., Bilgrami, A. L., Shapiro-Ilan, D., & Gaugler, R. (2007) Stability of entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus luminescens, during in vitro culture. Journal of Industrial Microbiology and Biotechnology , 34 (1), 73-81 Wood W, Gore J, Catchot A, Cook D, Dodds D, Krutz LJ (2016) Susceptibility of flowering cotton to damage and yield loss from tarnished plant bug (Hemiptera: Miridae). J Econ Entomol 109:1188–1195 Zhang X, Machado RA, Doan CV, Arce CC, Hu L, Robert CA (2019) Entomopathogenic nematodes increase predation success by inducing cadaver volatiles that attract healthy herbivores Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4529008","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":314919793,"identity":"2dd0608f-55b7-4e07-b5ff-af1866cb96a1","order_by":0,"name":"James P. Glover","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBACxgYILQMmPyAECGvhAXNmEKMFBsBamHmI0cLcfjrx4Q+GWh5+ieRjn23b6mT7G9gvPubB57Ce3M3GPAzHeSRnpCXPzm07bDzjAE+xMV4tDbnbpBkYjvEYnDljzJzbdiCx4QBPmuQMfFr6327/+QOs5fxnZsu2usT5BLXMyN0G9HsNj8HxHmZmxjbmxA0H2I9JfMCr5e1maR6DAzyS7W3GjD3nDhtvPMzDbIBPi2F/7saPPyrq5PiZmR8z/Cirk513vP3hgwR8WhpApMFhJCFmHgM8GhgY5CFUHbIY+wO8WkbBKBgFo2DEAQCwN02U8b8OLgAAAABJRU5ErkJggg==","orcid":"","institution":"United States Department of Agriculture","correspondingAuthor":true,"prefix":"","firstName":"James","middleName":"P.","lastName":"Glover","suffix":""},{"id":314919794,"identity":"471c1b37-1126-47b5-97e5-28089a491b0e","order_by":1,"name":"Nathan Spaulding","email":"","orcid":"","institution":"United States Department of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Nathan","middleName":"","lastName":"Spaulding","suffix":""},{"id":314919795,"identity":"921b738f-1797-499f-a45c-a0a630fbfc6e","order_by":2,"name":"Marissa I. Nufer","email":"","orcid":"","institution":"United States Department of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Marissa","middleName":"I.","lastName":"Nufer","suffix":""},{"id":314919796,"identity":"d3befb73-add0-4846-b734-25cbbfbe048a","order_by":3,"name":"Justin George","email":"","orcid":"","institution":"United States Department of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Justin","middleName":"","lastName":"George","suffix":""},{"id":314919797,"identity":"b1b81492-90c5-46bc-877e-d7958339abd9","order_by":4,"name":"Maribel Portilla","email":"","orcid":"","institution":"United States Department of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Maribel","middleName":"","lastName":"Portilla","suffix":""},{"id":314919798,"identity":"e4b7e38c-927c-4787-98a6-6ef4572aeb07","order_by":5,"name":"Gadi V.P. Reddy","email":"","orcid":"","institution":"United States Department of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Gadi","middleName":"V.P.","lastName":"Reddy","suffix":""}],"badges":[],"createdAt":"2024-06-04 15:06:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4529008/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4529008/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58827897,"identity":"d361de48-43e6-42e7-a14e-970354d94062","added_by":"auto","created_at":"2024-06-21 17:32:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":530291,"visible":true,"origin":"","legend":"\u003cp\u003eChoice arena\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4529008/v1/f4f6516c6176dcb5d4ddf7d6.png"},{"id":58827989,"identity":"6a9232f1-6ac4-4cc4-b99a-3437ed95b833","added_by":"auto","created_at":"2024-06-21 17:32:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":72468,"visible":true,"origin":"","legend":"\u003cp\u003eMortality (mean ± SE) of \u003cem\u003eL. lineolaris\u003c/em\u003e topically treated with a water control and ten EPN strains; Hg, Hb-HP88, Hb-HVS, Sra, Sf, Sc-Cxrd, Sri-355, Sc-A11, Sri-712, and Hf. Bars with different letters indicate significant differences (p \u0026lt; 0.05) between treatments by model contrast analysis based on a generalized linear mixed effect model.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4529008/v1/b7de2a3a7c237af510db72f5.png"},{"id":58827890,"identity":"22e9978a-6c30-498f-b9d6-6119b82b14e5","added_by":"auto","created_at":"2024-06-21 17:32:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72267,"visible":true,"origin":"","legend":"\u003cp\u003eMortality (mean ± SE) of \u003cem\u003eN. roseipennis\u003c/em\u003e topically treated with a water control and ten EPN strains; Hg, Hb-HP88, Hb-HVS, Sra, Sf, Sc-Cxrd, Sri-355, Sc-A11, Sri-712, and Hf. Bars with * indicate significant differences (p \u0026lt; 0.05) between treatments by model contrast analysis based on a generalized linear mixed effect model.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4529008/v1/a6c9eab8490089dd270118eb.png"},{"id":58828320,"identity":"1dd8960c-4e89-4462-acab-11f0cea15b4f","added_by":"auto","created_at":"2024-06-21 17:33:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":93436,"visible":true,"origin":"","legend":"\u003cp\u003eChoice predation response of \u003cem\u003eN. roseipennis\u003c/em\u003e to EPN \u003cem\u003eL. lineolaris \u003c/em\u003einfected with four EPN strains; Hb-HVS, Sra, Sf, Sc-Cxrd. * Indicate significant differences (p \u0026lt; 0.05) between treatments.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4529008/v1/9d4b8a960b7453e34a281ba6.png"},{"id":58828321,"identity":"d8536c55-4e44-46f2-85c4-b8ccae28e724","added_by":"auto","created_at":"2024-06-21 17:33:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":137948,"visible":true,"origin":"","legend":"\u003cp\u003ePercent predation by \u003cem\u003eN. roseipennis\u003c/em\u003e (mean ± SE) of \u003cem\u003eL. lineolaris \u003c/em\u003einfected with four EPN strains; Hb-HVS, Sra, Sf, Sc-Cxrd, and a water control. Bars with different letters indicate significant differences (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4529008/v1/6aec9907414996085627c7e0.png"},{"id":58828061,"identity":"ae15fa76-f682-4c33-ad76-9aab84bf5dfb","added_by":"auto","created_at":"2024-06-21 17:32:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1067733,"visible":true,"origin":"","legend":"\u003cp\u003eThird instar EPN infected tarnished plant bug showing characteristic reddish-brown coloration (a), Infected tarnished plant bug with abdomen dissected showing EPN proliferation (b), adults (larger) juvenile (arrow) (c).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4529008/v1/80b3b814d723108331ed4636.png"},{"id":75841747,"identity":"70e85830-5fe7-4c46-9e9e-2381f8307406","added_by":"auto","created_at":"2025-02-09 18:01:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2515495,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4529008/v1/f27c8af0-13c0-4008-81f5-24f3548f60e5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Infection, choice behavior, and cross infectivity of the sculpted damsel bug, Nabis roseipennis offered tarnished plant bug, Lygus lineolaris infected with entomopathogenic nematodes","fulltext":[{"header":"Key Message","content":"\u003cul\u003e\n \u003cli\u003eTrophic impacts of EPN\u0026apos;s on how predation is affected by infected prey has not been studied in detail\u003c/li\u003e\n \u003cli\u003eTarnished plant bugs were susceptible to all 10 EPN strains isolates within 72 hours\u003c/li\u003e\n \u003cli\u003eSculpted damsel bugs fed on infected prey significantly shorter times than healthy prey items\u003c/li\u003e\n \u003cli\u003eGeneralist predator never became infected with EPN\u0026rsquo;s despite feeding duration\u003c/li\u003e\n \u003cli\u003eTarnished Plant bug infected with nematodes are less attractive to predatory bugs aiding in biological control\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe tarnished plant bug, \u003cem\u003eLygus lineolaris\u003c/em\u003e (Palisot de Beauvois), is a major pest of row crops in the southern United States (Williams, 2015). Highly polyphagous, it feeds on a wide range of host plants, including the most economically important crops grown in the Midsouth (George et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Parys \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The tarnished plant bug has been recognized as a major economic pest of cotton, causing significant fruit loss and economic damage in cotton-producing regions across the mid-South, the Mississippi Delta region, and other areas of the world (Snodgrass et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Pest status is exacerbated by a multivoltine life cycle.\u003c/p\u003e \u003cp\u003eThe sculpted damsel bug, \u003cem\u003eNabis roseipennis\u003c/em\u003e Reuter (Hemiptera: Nabidae), is among the most abundant and most common of the damsel bugs encountered in cotton and soybean across the Southeastern United States (Pfannenstiel et al. 1998; Irwin et al. 1980; Shepard et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Pitre et al. 1978; and Dinkins et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1970\u003c/span\u003e). A complex of Nabids \u003cem\u003espp\u003c/em\u003e. are commonly found in traditional row crop agroecosystems, including cotton, soybean, and sorghum, and are predators of many Lepidopteran eggs, aphids, and other small soft-bodied insects (Coscar\u0026oacute;n et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Stewart et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Nymphal stages of many Nabid \u003cem\u003espp.\u003c/em\u003e can typically consume one or more eggs and or aphids per day, while more mature instars and adults may consume as many as two dozen prey items (Conti et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Haseeb et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; and Bueno et al. 2012). Many species of Nabids irrespective of nymphal growth stage have been reported to feed on all life stages of Lygus \u003cem\u003espp.\u003c/em\u003e (Perkins and Watson \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1972\u003c/span\u003e; Dumont et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Perkins et al. 1971).\u003c/p\u003e \u003cp\u003eEntomopathogenic nematodes (EPN's) are a group of parasitic nematodes that have been extensively studied for their potential use in the biological control of insect pests as well as a model for studying reproductive biology (Mahmoud, 2017). EPNs are obligate pathogens of insects and important biological control agents of insect pests (Pilz et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). EPN's in the genera \u003cem\u003eSteinernema\u003c/em\u003e and \u003cem\u003eHeterorhabditis\u003c/em\u003e, have shown great potential for biological pest control in specific agricultural settings. These nematodes are capable of infecting and killing insect pests, making them an attractive alternative to chemical pesticides, which can have negative effects on the environment and human health (Kaya and Gaugler, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). The use of nematodes as biological control agents has been widely researched, and EPNs have emerged as promising candidates (Kaya and Stock, 1997). EPN's have been found to be highly effective in controlling a variety of insect pests, including some of the most destructive pests in agriculture, such as the corn rootworm \u003cem\u003eDiabrotica spp.\u003c/em\u003e and black vine weevil \u003cem\u003eOtiorhynchus sulcatus\u003c/em\u003e (Toepfer et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Lewis et al. 2005). In addition, some species of EPN 's have been found to be highly host-specific, which makes them ideal for use as biological control agents since they are believed to cause less off-target effects on organisms (Gaugler and Kayla 1990; Abd-Elgawad, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Many species of EPN's have been shown to be effective against a wide range of insect pests, including white grubs, root weevils, and cutworms (Kaya and Gaugler, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). They have also been used successfully against soil-dwelling pests such as corn rootworms and wireworms (Stock and Goodrich-Blair, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The use of EPN's has been shown to reduce insect pest populations and increase crop yields in specific agricultural settings (Kaya and Gaugler, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Recently, Steenman et al. (2023) found that the EPN \u003cem\u003eS. carpocapsae\u003c/em\u003e was an effective biological control agent against three common mirid pest species within a greenhouse environment. Furthermore, the Potential of \u003cem\u003eNabis americoferus\u003c/em\u003e as a biological control agent of \u003cem\u003eLygus lineolaris\u003c/em\u003e in strawberry fields has recently been reported (Dumont et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, EPN's have several advantages over traditional chemical insecticides. Nematodes are highly host-specific to their target pests, having considerably less off-target or non-target effects on other organisms, leaving beneficial insects unharmed (Stock and Goodrich-Blair, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kaya and Gaugler, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1993\u003c/span\u003e.). They also have a short persistence in the environment, as they are vulnerable to desiccation and predation, which reduces the risk of accumulation in the food chain (Kaya and Gaugler, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). In addition, they do not pose a risk of developing resistance in pest populations (Stock and Goodrich-Blair, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Nematodes are less likely to develop resistance to chemical insecticides, which can become less effective over time as pests evolve resistance. Furthermore, nematodes are environmentally friendly and do not leave harmful residues in the soil or water.\u003c/p\u003e \u003cp\u003eThe objectives of these experiments were to assess the infectivity and efficacy of two groups of entomopathogenic nematodes (EPN), \u003cem\u003eSteinernema\u003c/em\u003e and \u003cem\u003eHeterorhabditis\u003c/em\u003e by direct topical exposure against the tarnished plant bug and the generalist cotton predator, the sculpted damsel bug. Determine if predator prey-choice is affected by EPN infection, and if feeding on EPN infected tarnished plant bug (TPB) prey items could result in a cross-infection of the predator. The goal of this research is to determine the extent to which EPNs could infect and cause mortality in hosts and identify any strain specific differences. To gain further insight into the practical application and potential of EPNs to control TPB in a production setting, we conducted additional greenhouse experiments to further analyze potential cross-infectivity and predation of an infected prey item by a generalist predator.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInsect source and rearing\u003c/h2\u003e \u003cp\u003eAll studies were conducted at the USDA-ARS Southern Insect Management Research Unit, Stoneville, MS. Laboratory-reared TPB and nabid colonies were established from field collections of mixed-age late instar nymphs and adults collected from primarily large stands of seedling Johnsongrass (\u003cem\u003eSorghum halepense\u003c/em\u003e) and supplemented with collections from other broadleaf weeds common across the Mid-south including Palmer amaranth (\u003cem\u003eAmaranthus palmeri\u003c/em\u003e). TPB colonies were reared on a semi-solid autoclavable diet routinely used for the mass rearing of \u003cem\u003eLygus lineolaris\u003c/em\u003e (Portilla et al. 2011). Nabids were reared individually to adulthood in an environmental chamber (Percival Scientific Inc, Perry, Iowa) at a temperature of 27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C, 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH, and a 14:10 (L:D) h photoperiod. Individuals were supplied with a 42 ml plastic cup containing a small piece of cotton saturated with a 2% sucrose solution as a water source and freshly hatched \u003cem\u003eHelicoverpa zea\u003c/em\u003e (Boddie) neonates as a live food source.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEPN cultivation and insect inoculation\u003c/h2\u003e \u003cp\u003eThe twenty EPN isolates were reared in the last instar larvae of the greater wax moth, \u003cem\u003eGalleria mellonella\u003c/em\u003e L. (Lepidoptera: Pyralidae), which were obtained from Josh's Frogs (Owosso, Michigan) (Table\u0026nbsp;1). The ratio of IJs to a host was 10 IJs/host. 200 IJs in 2 ml of aqueous suspension were pipetted to a 100 x 15 mm Petri dish with a single layer of VWR filter paper and 20 hosts. The petri dishes were then covered with a black plastic bag to protect them from light and incubated at room temperature (~\u0026thinsp;22\u0026deg;C). After the hosts died from EPN infection, the cadavers were moved to White traps (Kaya and Stock, 1997) that were also kept at room temperature and coved with a black plastic bag. The traps were checked every day for the emergence of IJs, which were collected and stored in tissue culture flasks in a 13\u0026deg;C refrigerator.\u003c/p\u003e \u003cp\u003eThe concentration of infective juvenile was counted using a phase hemacytometer (Hausser Scientific #1475, Waltham, Massachusetts) and viewed under a Leica S9 stereo microscope (Leica Microsystems, Wetzlar, Germany) and adjusted by adding deionized water to attain a final concentration of 200 IJs per/ml. Aqueous nematode solutions of 200 IJs per 1ml of deionized water were pipetted directly onto the pronotum and mesothoracic regions of both adult sculpted damsel bugs and one-day old 3rd instar nymph tarnished plant bugs, respectively. Individuals were observed under a light microscope by removing the plastic lid from the 42 ml plastic assay cup containing diet and were examined for externally visible infections with any stage of EPN. After the duration of the experiment (10d) cadavers were retained and placed in cultivation flasks to incubate and determine EPN infection status.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eList\u003c/b\u003e/codex of entomopathogenic species of nematodes (EPN's) used in a series of experiments presented.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEPN species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEPN origin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEPN strain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCodex\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHeterorhabditis georgiana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeorgia, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKesha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHeterorhabditis bacteriophora\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUtah, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHP88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHb-HP88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHeterorhabditis bacteriophora\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeorgia, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHVS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHb-HVS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSteinernema rarum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCombined from isolates found in Louisiana, USA and Mississippi, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17\u003cem\u003ec\u0026thinsp;+\u0026thinsp;e\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSra\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSteinernema feltiae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFrance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSf\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSteinernema carpocapsae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArkansas, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCxrd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSc-Cxrd\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSteinernema riobrave\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTexas, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e355\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSri-355\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSteinernema carpocapsae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeorgia, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSc-A11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSteinernema riobrave\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTexas, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u0026ndash;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSri-712\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHeterorhabditis floridensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeorgia, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHf\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eLab and greenhouse prey preference trials\u003c/h2\u003e \u003cp\u003ePredator preference assays were designed according to Avery et al. (2022), in brief, prey preference behavior of the SDB adults was studied using choice arenas made from\u003c/p\u003e \u003cp\u003esterile Petri dishes (150 x 15 mm: Fisher Scientific, Inc. Waltham, MA, USA) lined with a round VWR\u0026reg; Grade 410 qualitative filter paper, (VWR-Avantor, Radnor, PA, USA) and trimmed to fit within the circumference of the bottom petri dish securely leaving no gaps. Arenas were designed with a central release zone centered on the drawn line using the cap from a 50 ml falcon plastic centrifuge tube placed in the center of each arena.\u003c/p\u003e \u003cp\u003eThird instar tarnished plant bugs were topically treated with aqueous nematode solutions of 200 IJs per 1ml of deionized water were pipetted directly onto the pronotum and mesothoracic regions, held on green beans sections (15mm) previously triple washed with 10% NaClO, in petri dishes (52 x 5 mm) lined with a round filter paper of similar size for 24h before being offered to SDB adults. One live 24h infected tarnished plant bug and one live uninfected nymph (water control) were placed opposite and perpendicular to the principal axis line. Tarnished plant bug nymphs were brushed with a natural hair paint brush (#0) and placed at equal distances from the drawn line (12.7 mm). Featherweight forceps were used to transfer SDB adults from rearing cups to under the 50ml falcon tube cap with a circular hole punched (20mm) in the center (Fig.\u0026nbsp;1). Plastic infesting caps were removed once the SDB adult exited the hole in the cap and was freely moving in the arena on the filter paper. Petri dish choice assays were performed in a walk-in environmental chamber with 16: 8 h (L:D) photoperiod, 28\u0026deg;C, and 15% RH with arenas placed directly under overhead luminescent light. Predator preference trials resulted in one of three possible outcomes: feeding on an infected TPB prey, feeding on an uninfected TPB nymph, or no choice. A \"predation event\" was defined as a prolonged stylet insertion and feeding event lasting\u0026thinsp;\u0026ge;\u0026thinsp;60 seconds. Preference trials commenced and lasted 20 min once the SDB exited from the hole in the cap and made contact with the filter paper. Trial replicates received freshly cleaned and air-dried Petri dishes and caps (75% alcohol) supplied with a new filter paper. The orientation of the infected and uninfected prey types was randomly assigned to exclude any directional bias.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGreenhouse experiments were performed on individual non-Bt cotton plants (DLP1892, delta pine, ark, USA) in two-gallon size black nursery greenhouse pots in standard potting media. SDB were released onto greenhouse-grown cotton in the second week of blooming (approx. 12 nodes). Cotton terminals were isolated with insect enclosure bags constructed from organza (22 x 22 x 8 mm\u0026thinsp;~\u0026thinsp;240 \u0026micro;m mesh, JoAnn\u0026rsquo;s Fabrics, Hudson, OH) that enclosed the plant terminals affixed with pipe cleaners (top five nodes) containing many squaring sites. The second week of bloom (\u0026asymp;\u0026thinsp;55d) was chosen to reflect not only a physiologically vulnerable growth stage of cotton but also a location that's commonly associated with economic damage. We conducted 20 replicates for the strains with mortality\u0026thinsp;\u0026gt;\u0026thinsp;70% (strains Hb-HVS, Sra, Sf, Sc-Cxrd) (Fig.\u0026nbsp;2.). Plants were infested by carefully opening the enclosure and paint brushing a 24h infected TPB 3rd instar nymphs, one per cage, onto a fully mature and opened terminal leaf and allowing 10 min to acclimate and explore the canopy. Featherweight forceps were used to transfer SDB adults from rearing cups to caged plant terminals, placed on an opposing mature leaf, and the cage securely fastened after the TPB acclimation period. Cages were vigorously inspected to ensure insects were not immobilized by the folds in the fabric and were completely confined to the desired nodes of growth. Cages were inspected every 24h for predated TPB as evidenced by a shriveled and imploded body indicative of feeding similar to individuals fed on during the petri dish assays. Many cages inspected were observed with SDB actively feeding on or actively roaming and carrying the TPB nymph.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eAll experimental measurements of mortality and infection were analyzed as a randomized complete block with a factorial arrangement in PROC GLM (Littell et al. 1991) (10 EPN strains examined x 3rd instar tarnished plant bug and adult sculpted damsel bugs exposed plus a water control). Each treatment combination was repeated three times. A Tukey's mean separation test (α\u0026thinsp;=\u0026thinsp;0.05) was used to compare results. Individuals who made no prey choice or who never excited the cap were excluded from the analysis. Predator preferences and time data (min) between infected and uninfected prey were analyzed by using a \u003cem\u003eX\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e test of independence to estimate the difference in probability of choosing infected versus control for each strain at each time point, allowing time to be treated as a continuous variable with non-linear time trend.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eInfection and mortality of tarnished plant bug nymphs\u003c/h2\u003e \u003cp\u003eThird instar tarnished plant bug mortality at seven days was significantly affected by topical exposure with the ten EPN strains (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;93.69; df\u0026thinsp;=\u0026thinsp;10, 418; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with mortality ranging from 57\u0026ndash;93%. The EPN strains with the highest mortality observed across the study were Sra and Sc-Cxrd with 94%, 92%, respectively (Fig.\u0026nbsp;2). No significant interactions were detected (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) across this study. A Tukey's post hoc means separations test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (α\u0026thinsp;=\u0026thinsp;0.05) determined that strains Hb-HP88, Hb-HVS, SRA, SF, and Sc-Cxrd caused significant mortality at five and seven days post topical exposure, and significant mortality on day three for Sri-355 when compared to strains Sc-A11, Sc-A11, HF, and controls (Fig.\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eInfection and mortality of adult nabids\u003c/h2\u003e \u003cp\u003eAdult nabid mortality one-week post exposure to the ten EPN strains was significantly higher for strains Sc-Cxrd and Sc-A11 treatments, and the highest mortality observed ranging from 27% -38%, respectively (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.19; df\u0026thinsp;=\u0026thinsp;10, 316; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;3). Significant interactions of main effects were not detected (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). A Tukey's post hoc means separations test (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (α\u0026thinsp;=\u0026thinsp;0.05) determined that nabids exposed to strain Sc-Cxrd experienced significant mortality at all three sampling points and on day seven for strain Sc-A11 post exposure (Fig.\u0026nbsp;3). All strains caused mortality\u0026thinsp;\u0026gt;\u0026thinsp;5% excluding strains Hb-HP88, Hb-HVS, SRA, and HF where no activity was detected.\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003ePredator preference\u003c/h2\u003e \u003cp\u003eIn the arena choice assays where nabids had a choice of uninfected tarnished plant bug prey item versus infected with strain Hb-HVS, 59.4% of individuals made no choice (n\u0026thinsp;=\u0026thinsp;69). 32.8% of nabids fed on the control tarnished plant bug (topical water exposure) (n\u0026thinsp;=\u0026thinsp;38), and 7.8% (n\u0026thinsp;=\u0026thinsp;9) fed on an infected prey item (Fig.\u0026nbsp;4). Exposed and infected tarnished plant bug prey were attacked significantly less by adult nabids compared to uninfected prey. On average nabids consumed infected prey items for 45 s\u0026thinsp;\u0026plusmn;\u0026thinsp;13 s (range 30\u0026ndash;75 s) compared to 321\u0026thinsp;\u0026plusmn;\u0026thinsp;17 s (range 60 \u0026minus;\u0026thinsp;900 s) on the uninfected prey \u003cem\u003ex\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e(1, \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;116)\u0026thinsp;=\u0026thinsp;11.84, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0006. Nabids that received tarnished plant bug prey items infected with strain Sra or a control insect made no choice 56% (n\u0026thinsp;=\u0026thinsp;105), while 40.6% (n\u0026thinsp;=\u0026thinsp;76) fed on the control tarnished plant bug, and 3.2% (n\u0026thinsp;=\u0026thinsp;6) fed on infected prey item (Fig.\u0026nbsp;4). Exposed and infected tarnished plant bug prey were attacked significantly less \u003cem\u003ex\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e(1, \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;187)\u0026thinsp;=\u0026thinsp;13.55, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0002 by adult nabids compared to uninfected tarnished plant bugs. The average time for a nabid to choose and feed on the EPN infected tarnished plant bug prey was 30 s, compared to 452\u0026thinsp;\u0026plusmn;\u0026thinsp;25 s (range 12\u0026ndash;900 s) for the nabids that chose the uninfected prey. Nabids, when offered a choice of infected, tarnished plant bug prey infected with strain SF or control, 49% nabids made no choice (n\u0026thinsp;=\u0026thinsp;92), 33% fed on the control tarnished plant bug (n\u0026thinsp;=\u0026thinsp;62), and 17% (n\u0026thinsp;=\u0026thinsp;33) fed on infected prey item (Fig.\u0026nbsp;4). Exposed and infected tarnished plant bug prey were attacked significantly less by adult nabids compared to uninfected prey. The average time for a nabid to choose and feed on the EPN infected tarnished plant bug prey was 153 s, compared to 565\u0026thinsp;\u0026plusmn;\u0026thinsp;15 s (range 12\u0026ndash;900 s) for the nabids that chose the uninfected prey \u003cem\u003ex\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e(1, \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;187)\u0026thinsp;=\u0026thinsp;14.72, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. Nabids that were given tarnished plant bug prey items infected with Sc-Cxrd on an uninfected prey item had 39% (n\u0026thinsp;=\u0026thinsp;73) of individuals made no choice, 44% (n\u0026thinsp;=\u0026thinsp;82) feed on the control tarnished plant bug (topical water exposure), and 17% (n\u0026thinsp;=\u0026thinsp;32) fed on an infected prey item. Exposed and infected tarnished plant bug prey were attacked significantly less by adult nabids compared to uninfected prey. The average time for a nabid to choose and feed on the Sc-Cxrd EPN infected tarnished plant bug prey was 62 s, compared to 528\u0026thinsp;\u0026plusmn;\u0026thinsp;37 s (range 25\u0026ndash;900 s) for the nabids that chose the uninfected prey \u003cem\u003ex\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e(1, \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;187)\u0026thinsp;=\u0026thinsp;11.63, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0006. Longest feeding of nabids on EPN infected tarnished plant bugs was observed with strain SF and strain Sc-Cxrd for 204 s and 180 s, respectively (Fig.\u0026nbsp;4).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGreen house no choice assay and cross-infectivity\u003c/h2\u003e \u003cp\u003eNabids individually infested on greenhouse-grown cotton plants consumed\u0026thinsp;\u0026gt;\u0026thinsp;80% of infected prey items in caged no-choice assays across the four strains tested (Fig.\u0026nbsp;5). Of the nabids who made a choice to feed on infected tarnished plant bug prey (n\u0026thinsp;=\u0026thinsp;81) none of the individuals regardless of the duration of the feeding event become infected with any of the four EPN strains tested in this experiment (Table\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eFirst discovered in the early 1920s, interest in EPNs increased in the 1950s with commercialization of the fist species in the 1980s. Given the safety profile to humans, the environment, and non-target species EPNs have been excluded from pesticide registration hurdles (Ehlers \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Piedra-Buena et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Research on the application, biology, and ecology of EPNs began in the early1990s and further expanded into basic research in the 2000s. The extensive body of literature resulting from this research has resulted in several seminal books (Gaugler and Kaya, 1990; Bedding et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Gaugler, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Grewal et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Nabidae mouthpart or rostrum is composed of four flexible and mobile segments that sets at the mesocoxae at resting position. The first two joints are thick and rectilinear and are the most flexible followed by the third and generally longest segment (Pericart 1987). The relationship between nematodes specifically \u003cem\u003eSteinernema\u003c/em\u003e and \u003cem\u003eHeterorhabditis\u003c/em\u003e and their bacterial symbionts \u003cem\u003eXenorhabdus\u003c/em\u003e spp. and \u003cem\u003ePhotorhabdus\u003c/em\u003e spp., respectively are highly specific (Shaprio-llan et al. 2023). These bacteria are the primary causative agent responsible for killing the host insect and providing nutrition for the nematodes (Lewis \u0026amp; Clarke \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e is a bacterial insect pathogen from the Enterobacteriaceae family of soil-based nematodes from the genera \u003cem\u003eHeterorhabditis\u003c/em\u003e. This bacterium produces an array of toxins that assemble as a complex (approximately 25 nm in length and 1.5 nm in diameter) (Alouf et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). EPN associated \u003cem\u003eXenorhabdus\u003c/em\u003e spp. bacteria have been reported to range in size approximately 2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 \u0026micro;m\u003csup\u003e3\u003c/sup\u003e (Wang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The average size of the smallest and forth section of the sculpted damsel bug was approximately 0.6 mm and 0.021 length and diameter, respectively (Glover unpublished data). Given the size of the symbiotic bacteria and the relative mouthpart size of the damsel bug it is theoretically possible for the microbes to be acquired. However, acquisition and transmission of EPN bacterial symbionts from the Enterobacteriaceae family remains unknown. Given the longevity of adult \u003cem\u003eNabis\u003c/em\u003e spp., which has been reported to be almost 60 days (Rebolledo et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), there is a dearth of information concerning its interactions with EPN's. So far, the trophic impact of EPN's on how predation is affected by infected prey items has not been studied in detail. The experimental data presented in this study is the first to investigate EPN associated mortality and feeding behavior of the sculpted damsel bug offered EPN infected prey items.\u003c/p\u003e \u003cp\u003eOur data shows that the tarnished plant bug was susceptible to all ten EPN strains tested in the present study with mortality rates of more than 60% reached after seven days. However, strains Hb-HP88, Hb-HVS, Sra, Sf, and Sc-Cxrd showed marked mortality\u0026thinsp;\u0026gt;\u0026thinsp;50% three days post-exposure to infective juveniles. Similarly, Steenman et al. (2023) found that three common mirid pest species were similarly susceptible to the EPN family \u003cem\u003eS. carpocapsae\u003c/em\u003e (ScAll and ScCxrd). Many of the infected tarnished plant bug nymphs began to change body coloration to reddish-brown as infection progressed and may be a reliable indicator of EPN progressive infection (Fig.\u0026nbsp;6). Mortality for the sculpted damsel bug was significantly affected by strains Sc-Cxrd and strain Sc-A11 with mortality ranging from 25\u0026ndash;38% respectively. Conversely, strains Hb-Hp88, Hb-HVS, Sra, and Hf had no significant effect on mortality. Based on our data, we choose strains Hb-HVS, Sra, SF, and Sc-Cxrd due to the high associated mortality on the tarnished plant bug and the relatively low or no mortality associated with the sculpted damsel bug. This selection allowed for the testing of four strains from the two major genus of EPN currently under investigation in modern agriculture. Our study indicates that the sculpted damsel bug primarily avoided EPN infected prey items when given a choice to predate a healthy prey item, and greater avoidance was observed after an individual made contact first with an infected prey item. Damsel bugs fed on infected prey items (81) across the entirety of the experiment using four strains tested. However, we found that no damsel bug had been infected with any strain or life stage of EPN across the strains tested here after consumption, regardless of the feeding duration of an infected EPN tarnished plant bug prey item.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of this study may also provide insight into the general effects of EPNs on predator-prey choice. In this study, our generalist predator, the sculpted damsel bug, actively fed on and probed infected prey items for significantly shorter times than uninfected or healthy prey when offered a choice. The sculpted damsel generally avoided the infected tarnished plant bug, particularly after feeding and probing contact with an infected individual in choice arenas. Of the 81 instances where the predator fed on the infected prey item, more than half of the feeding events lasted for \u0026lt;\u0026thinsp;60 seconds, and the predator actively avoided infected prey in choice of the healthy prey item for the remainder of the assay. Cadavers hosting the nematode/bacteria complex have been shown to be highly deterrent for detritivorous insects and was fist demonstrated with the EPN \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e (Heterorhabditidae) and workers of \u003cem\u003eLinepithema humile\u003c/em\u003e (Hymenoptera: Formicidae) (Baur et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Mertz et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) demonstrated that the predator \u003cem\u003eCalosoma granulatum\u003c/em\u003e actively avoided feeding on infected larvae of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e when given a choice of healthy versus infected prey types. The sculpted damsel bug generally avoided the infected prey items in the choice experiments presented here. This observation may translate to field avoidance of EPN infected prey. It may further facilitate an even greater number of predation events of uninfected prey, leading to increased predation of tarnished plant bugs in a field setting. However, greenhouse assays demonstrated that under no-choice conditions that predators would actively search and predate infected tarnished plant bugs\u0026thinsp;\u0026gt;\u0026thinsp;75% with some avoidance observed, however no predators ever become externally or internally infected with any stage of EPN strain tested.\u003c/p\u003e \u003cp\u003eThe results obtained in the present experiments show that in general, the of the ten EPN isolates Hb-HVS, Sra, SF, and Sc-Cxrd have high potential for use in biological control against the TPB, are safe for the native generalist predator \u003cem\u003eN. roseipennis.\u003c/em\u003e Further investigation in a field setting will provide additional behavioral information on how potential volatile bouquets influence prey visitation and predation events (Zhang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In conclusion, the use of EPN's as a biological control method in agriculture shows great promise. They are effective against a wide range of insect pests, specific to their target pests, and have several advantages over chemical pesticides. As such, further research on the development of encapsulation and U.V. protectant technologies will increase the efficiency of biological control of pests in an integrated pest management (IPM) system.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by James P. Glover, Justin George and Gadi V.P. Reddy. The first draft of the manuscript was written by James Paul Glover and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e Research involving human participants and/or animals, Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish:\u003c/strong\u003e Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbd-Elgawad MM (2019) Towards optimization of entomopathogenic nematodes for more service in the biological control of insect pests. Egypt J Biol Pest Control 29:1\u0026ndash;8\u003c/li\u003e\n \u003cli\u003eAlouf, J. E., Ladant, D., \u0026amp; Popoff, M. R. (2005) \u003cem\u003eThe comprehensive sourcebook of bacterial protein toxins\u003c/em\u003e. Elsevier\u003c/li\u003e\n \u003cli\u003eBaur ME, Kaya HK, Strong DR (1998) Foraging ants as scavengers on entomopathogenic nematode-killed insects. Biol Control 12:231\u0026ndash;236\u003c/li\u003e\n \u003cli\u003eBedding, R. A., Akhurst, R. J., \u0026amp; Kaya, H. K. (1993) \u003cem\u003eNematodes and the biological control of insect pests\u003c/em\u003e. Csiro Publishing. pp 1350-1356.\u003c/li\u003e\n \u003cli\u003eBraman SK, Yeargan KV (1990) Phenology and abundance of \u003cem\u003eNabis americoferus\u003c/em\u003e, \u003cem\u003eN. roseipennis\u003c/em\u003e, and \u003cem\u003eN. rufusculus\u003c/em\u003e (Hemiptera: Nabidae) and their parasitoids in alfalfa and soybean. J Econ Entomol 83:823\u0026ndash;830\u003c/li\u003e\n \u003cli\u003eBueno VHP, van Lenteren JC (2012) Predatory bugs (Heteroptera), in \u003cem\u003eInsect Bioecology and Nutrition for Integrated Pest Management\u003c/em\u003e, ed. by AR Panizzi and J Parra. CRC Press, Boca Raton, FL, pp. 539\u0026ndash;569\u003c/li\u003e\n \u003cli\u003eBurnell, A., \u0026amp; Stock, S. P. (2000) Heterorhabditis, Steinernema and their bacterial symbionts\u0026mdash;lethal pathogens of insects. \u003cem\u003eNematology\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e: 31-42\u003c/li\u003e\n \u003cli\u003eCampbell JF, Gaugler R, (1993) Entomopathogenic nematodes: a review of biology, behavior and host-parasite interactions. J Nematol 25:235\u0026ndash;249\u003c/li\u003e\n \u003cli\u003eCampos\u0026ndash;Herrera, R., Barbercheck, M., Hoy, C. W., \u0026amp; Stock, S. P. (2012) Entomopathogenic nematodes as a model system for advancing the frontiers of ecology. \u003cem\u003eJournal of nematology\u003c/em\u003e, \u003cem\u003e44\u003c/em\u003e:162\u003c/li\u003e\n \u003cli\u003eCatchot A, Musser F, Gore J, Cook D, Daves C, Lorenz G, Studebaker G (2009) Midsouth Multistate Evaluation.\u003c/li\u003e\n \u003cli\u003eConti E, Avila G, Barratt B, Cingolani F, Colazza S, Guarino S, Wajnberg E (2021) Biological control of invasive stink bugs: Review of global state and future prospects. Entomol Exp Appl 169:28\u0026ndash;51\u003c/li\u003e\n \u003cli\u003eCoscar\u0026oacute;n MC, Braman SK, Cornelis M (2015) Damsel Bugs (Nabidae). In: Panizzi, A., Grazia, J. (eds) True Bugs (Heteroptera) of the Neotropics. Entomology in Focus, vol 2. Springer, Dordrecht. https://doi.org/10.1007/978-94-017-9861-7_11\u003c/li\u003e\n \u003cli\u003eDaoust, SP, King KC, Brodeur J, Roitberg BD, Roche B, Thomas F (2015) Making the best of a bad situation: host partial resistance and bypass of behavioral manipulation by parasites? Trends Parasitol 31:413\u0026ndash;418\u003c/li\u003e\n \u003cli\u003eDinkins RL, Brazzel JR, Wilson CA (1970) Seasonal incidence of major predaceous arthropods in Mississippi cotton fields. J Econ Entomol 63:814\u0026ndash;817\u003c/li\u003e\n \u003cli\u003eDumont, F., Sol\u0026agrave;, M., Provost, C., \u0026amp; Lucas, E. (2023) The potential of Nabis americoferus and Orius insidiosus as biological control agents of Lygus lineolaris in strawberry fields. \u003cem\u003eInsects\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(4), 385.\u003c/li\u003e\n \u003cli\u003eEhlers, R. U. (2005) Forum on safety and regulation: Nematodes as biocontrol agents. Wallingford UK: CABI publishing, 107-114\u003c/li\u003e\n \u003cli\u003eGaugler, R. (1990) Entomopathogenic nematodes in biological control (Vol. 227). H. K. Kaya (Ed.). Boca Raton: CRC press\u003c/li\u003e\n \u003cli\u003eGaugler, R. (2002) Entomopathogenic Nematology. Wallingford, UK: CABI Publishing. doi: 10.1079/9780851995670.0000\u003c/li\u003e\n \u003cli\u003eGeorge J, Glover JP, Gore J, Crow WD, Reddy GV (2021) Biology, ecology, and pest management of the tarnished plant bug, \u003cem\u003eLygus lineola\u003c/em\u003eris (Palisot de Beauvois) in southern row crops. Insects 12:807\u003c/li\u003e\n \u003cli\u003eGodfrey KE, Whitcomb WH, Stimac JL (1989) Arthropod predators of velvetbean caterpillar, \u003cem\u003eAnticarsia gemmatalis\u003c/em\u003e H\u0026Atilde;ubner (Lepidoptera: Noctuidae), eggs and larvae. Environ Entomol 18:118\u0026ndash;123\u003c/li\u003e\n \u003cli\u003eGrewal, P. S., Ehlers, R. U., \u0026amp; Shapiro-Ilan, D. I. (Eds.). (2005) \u003cem\u003eNematodes as biocontrol agents\u003c/em\u003e. CABI\u003c/li\u003e\n \u003cli\u003eHaseeb M, Gordon TL, Kanga LH, Legaspi JC (2018) Abundance of natural enemies of \u003cem\u003eNezara viridula\u003c/em\u003e (Hemiptera: Pentatomidae) on three cultivars of sweet alyssum. J Appl Entomol 142:847\u0026ndash;853\u003c/li\u003e\n \u003cli\u003eIrwin ME, Shepard M (1980) Sampling Predaceous Hemiptera on Soybean. In: Kogan, M., Herzog, D.C. (eds) Sampling Methods in Soybean Entomology. Springer Series in Experimental Entomology. Springer, New York, NY. https://doi.org/10.1007/978-1-4612-9998-1_25\u003c/li\u003e\n \u003cli\u003eKaya HK, Gaugler R (1993) Entomopathogenic nematodes. Ann Rev Entomol 38:181\u0026ndash;206.\u003c/li\u003e\n \u003cli\u003eLewis, E. E., \u0026amp; Clarke, D. J. (2012) Nematode parasites and entomopathogens. In \u003cem\u003eInsect pathology\u003c/em\u003e (pp. 395-424). Academic Press\u003c/li\u003e\n \u003cli\u003eLewis, E. E., \u0026amp; Grewal, P. S. (2005) Interactions with plant-parasitic nematodes\u003c/li\u003e\n \u003cli\u003eMertz, N. R., Agudelo, E. J. G., Sales, F. S., \u0026amp; Moino Junior, A. (2015) Effects of entomopathogenic nematodes on the predator \u003cem\u003eCalosoma granulatum\u003c/em\u003e in the laboratory. \u003cem\u003eJournal of insect behavior\u003c/em\u003e, \u003cem\u003e28\u003c/em\u003e, 312-327.\u003c/li\u003e\n \u003cli\u003eParys KA (2014) Host plants of the tarnished plant bug, \u003cem\u003eLygus lineolaris\u003c/em\u003e (Palisot de Beauvois). Beltwide Cotton Conferences, 6\u0026ndash;8 January 2014, National Cotton Council\u003c/li\u003e\n \u003cli\u003ePerkins, P. V., \u0026amp; Watson, T. F. (1972) Biology of Nabis alternatus (Hemiptera: Nabidae). \u003cem\u003eAnnals of the Entomological Society of America\u003c/em\u003e, \u003cem\u003e65\u003c/em\u003e(1), 54-57.\u003c/li\u003e\n \u003cli\u003eP\u0026eacute;ricart, J. (1987) \u003cem\u003eH\u0026eacute;mipt\u0026egrave;res Nabidae d\u0026apos;Europe occidentale et du Maghreb\u003c/em\u003e. F\u0026eacute;d\u0026eacute;ration fran\u0026ccedil;aise des soci\u0026eacute;t\u0026eacute;s de sciences naturelles\u003c/li\u003e\n \u003cli\u003ePilz C, Toepfer S, Knuth P, Strimitzer T, Heimbach U, Grabenweger G (2014) Persistence of the entomoparasitic nematode \u003cem\u003eHeterorhabditis bacteriophora\u003c/em\u003e in maize fields. J Appl Entomol 138:202\u0026ndash;212\u003c/li\u003e\n \u003cli\u003ePiedra-Buena, A., L\u0026oacute;pez-Cepero, J., \u0026amp; Campos-Herrera, R (2015) Entomopathogenic nematode production and application: regulation, ecological impact and non\u0026ndash;target effects. In Nematode Pathogenesis of Insects and Other Pests: Ecology and Applied Technologies for Sustainable Plant and Crop Protection (pp. 255-282). Cham: Springer International Publishing\u003c/li\u003e\n \u003cli\u003ePfannenstiel RS, Yeargan KV (1998) Ovipositional preference and distribution of eggs in selected field and vegetable crops by \u003cem\u003eNabis roseipennis\u003c/em\u003e (Hemiptera: Nabidae). J Entomol Sci 33:82\u0026ndash;89\u003c/li\u003e\n \u003cli\u003ePortilla, M., G. Snodgrass, and D. Streett (2010) Effect of modification of the NI artificial diet on the biological fitness parameters of mass reared western tarnished plant bug, Lygus hesperus. J. Insect Sci. 11: 1\u0026ndash;10.\u003c/li\u003e\n \u003cli\u003eRebolledo R, Villegas G, Klein C, Aguilera A (2005) Fluctuaci\u0026oacute;n poblacional, capacidad depredadora y longevidad de Nabis punctipennis Blanchard (Hemiptera: Nabidae). \u003cem\u003eAgric T\u0026eacute;cnica\u003c/em\u003e, 65:442\u0026ndash;446\u003c/li\u003e\n \u003cli\u003eShapiro-Ilan, D. I., Leite, L. G., \u0026amp; Han, R. (2023) Production of entomopathogenic nematodes. In \u003cem\u003eMass production of beneficial organisms\u003c/em\u003e (pp. 293-315). Academic Press\u003c/li\u003e\n \u003cli\u003eShapiro-Ilan, D., \u0026amp; Dolinski, C. (2015) Entomopathogenic nematode application technology. \u003cem\u003eNematode Pathogenesis of Insects and Other Pests: Ecology and Applied Technologies for Sustainable Plant and Crop Protection\u003c/em\u003e, 231-254\u003c/li\u003e\n \u003cli\u003eShapiro-Ilan DI, Gouge DH, Piggott SJ, Fife JP (2006) Application technology and environmental considerations for use of entomopathogenic nematodes in biological control. Biol Control 38:124\u0026ndash;133\u003c/li\u003e\n \u003cli\u003eShapiro-Ilan, D. I., Gouge, D. H., Koppenh\u0026ouml;fer, A. M., \u0026amp; Gaugler, R. (2002) Entomopathogenic nematology. \u003cem\u003eWallingford: CABI Publishing\u003c/em\u003e, 333-355\u003c/li\u003e\n \u003cli\u003eShepard M, Carner GR, Turnipseed SG (1974) Seasonal abundance of predaceous arthropods in soybeans. Environ Entomol 3:985\u0026ndash;988\u003c/li\u003e\n \u003cli\u003eSingh, A. K., Kumar, M., Ahuja, A., Vinay, B. K., Kommu, K. K., Thakur, S., ... \u0026amp; Parihar, M. (2022) Entomopathogenic nematodes: A sustainable option for insect pest management. In \u003cem\u003eBiopesticides\u003c/em\u003e (pp. 73-92). Woodhead Publishing\u003c/li\u003e\n \u003cli\u003eSnodgrass G, Gore J, Abel C, Jackson R (2009) Acephate resistance in populations of the tarnished plant bug (Heteroptera: Miridae) from the Mississippi River Delta. J Econ Entomol 102:699\u0026ndash;707\u003c/li\u003e\n \u003cli\u003eStewart SD, Layton B, Catchot A (2007) Common beneficial arthropods found in field crops. University of Tennessee Extension.\u003cem\u003e\u0026nbsp;\u003c/em\u003ehttp://msucares. com/pubs/publications/e0020. Pdf\u003c/li\u003e\n \u003cli\u003eStock SP, Goodrich-Blair H (2012) Entomopathogenic nematodes and their bacterial symbionts: the inside out of a mutualistic association. Symbiosis 58:47\u0026ndash;65\u003c/li\u003e\n \u003cli\u003eToepfer, S., Peters, A., Ehlers, R. U., \u0026amp; Kuhlmann, U. (2008) Comparative assessment of the efficacy of entomopathogenic nematode species at reducing western corn rootworm larvae and root damage in maize. \u003cem\u003eJournal of Applied Entomology\u003c/em\u003e, 132:337-348\u003c/li\u003e\n \u003cli\u003eWang, Y., Bilgrami, A. L., Shapiro-Ilan, D., \u0026amp; Gaugler, R. (2007) Stability of entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus luminescens, during in vitro culture. \u003cem\u003eJournal of Industrial Microbiology and Biotechnology\u003c/em\u003e, \u003cem\u003e34\u003c/em\u003e(1), 73-81\u003c/li\u003e\n \u003cli\u003eWood W, Gore J, Catchot A, Cook D, Dodds D, Krutz LJ (2016) Susceptibility of flowering cotton to damage and yield loss from tarnished plant bug (Hemiptera: Miridae). J Econ Entomol 109:1188\u0026ndash;1195\u003c/li\u003e\n \u003cli\u003eZhang X, Machado RA, Doan CV, Arce CC, Hu L, Robert CA (2019) Entomopathogenic nematodes increase predation success by inducing cadaver volatiles that attract healthy herbivores\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"tri-trophic interactions, predation, entomopathogenic nematodes, beneficial insects, crop pests","lastPublishedDoi":"10.21203/rs.3.rs-4529008/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4529008/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe tarnished plant bug, \u003cem\u003eLygus lineolaris\u003c/em\u003e Palisot de Beauvois (Hemiptera: Miridae), is an economically important pest of row crops worldwide. Ten isolates of entomopathogenic nematodes (EPNs) (Rhabditida: Steinernematidae and Heterorhabditidae) were evaluated against the third instar nymphal stage of tarnished plant bug and its generalist predator, the sculpted damsel bug, \u003cem\u003eNabis roseipennis\u003c/em\u003e Reuter (Hemiptera: Nabidae) one of the most abundant and commonly encountered damsel bugs in cotton and soybean agroecoscapes across the Southeastern United States. The objectives of these experiments were to assess the infectivity of entomopathogenic nematodes (EPN) by direct topical exposure against the sculpted damsel bug and tarnished plant bug, and whether the predator prey-choice is affected by EPN infection, and if feeding on EPN infected tarnished plant bug (TPB) prey items could result in cross-infection of the predator. Mortality rates at a concentration of 200 infective juveniles (IJs)/ml significantly differed among isolates and insect species, ranging from 30\u0026ndash;93% for tarnished plant bugs and sculpted damsels 6\u0026ndash;38%, respectively. The third instars of \u003cem\u003eL. lineolaris\u003c/em\u003e were more susceptible to the ten nematode isolates than \u003cem\u003eN. roseipennis\u003c/em\u003e. Higher pathogenicity on the tarnished plant bug and low mortality potential make strains HbHP88, HbVS, Sc17c\u0026thinsp;+\u0026thinsp;e, and SfSN the most promising candidate for the biological control of \u003cem\u003eL. lineolaris\u003c/em\u003e under lab and greenhouse conditions while preserving beneficial predators of the Southeastern United States.\u003c/p\u003e","manuscriptTitle":"Infection, choice behavior, and cross infectivity of the sculpted damsel bug, Nabis roseipennis offered tarnished plant bug, Lygus lineolaris infected with entomopathogenic nematodes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-21 16:15:20","doi":"10.21203/rs.3.rs-4529008/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c860497f-131e-429d-a03d-d6b2b8f1d8f5","owner":[],"postedDate":"June 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-09T17:53:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-21 16:15:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4529008","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4529008","identity":"rs-4529008","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}