The tomato leafminer, Phthorimaea absoluta is a serious insect pest that threatens worldwide tomato production. In the present study, the compatibility of two native Iranian entomopathogenic nematode (EPN) isolates, Steinernema feltiae IRSardrood and Steinernema carpocapsae IRMoghan1, combined with flubendiamide, a convenient bio-rational insecticide, was measured using Galleria mellonella as a surrogate host. In a separate experiment, the efficacy of the EPNs, flubendiamide, and their combinations against P. absoluta larvae were evaluated at two relative humidity (RH) regimes. Mortality of EPN isolates treated with flubendiamide did not differ significantly during the first 72 h; however, a pronounced increase was observed for S. carpocapsae IRMoghan1 at day 7. Bioassay results against P. absoluta larvae revealed that S. carpocapsae IRMoghan1 (83.83%) and flubendiamide (89.17%) caused higher mean larval mortality than S. feltiae IRSardrood (73.43%). Steinernema feltiae IRSardrood caused the highest insect mortality (99%) at RH >= 90% and the lowest mortality (47.87%) at 70% RH. In conclusions, the high survival rates of the flubendiamide-treated EPNs up to 72 h and no adverse effect on their pathogenicity. The high efficacies of the nematodes and nematode-insecticide combinations against P. absoluta larvae, indicate the high compatibility of these nematodes with flubendiamide, which outlines a promising perspective for the inclusion of the native EPN isolates in the integrated control of P. absoluta.

Entomopathogenic nematodes (EPNs) have a specialized infective juvenile stage (IJ) that is mobile and has the capability to seek insect hosts to penetrate their haemocoel. EPNs are primarily applied to soil as biological control agents; thus, the IJs must move through the soil to find and infect a host. Soil characteristics are known to be an important factor that can affect the efficiency of EPN movement behavior. Previous research has shown that exposure to ascaroside pheromones can enhance EPN movement and infectivity in soil. The ability of pheromones to enhance EPN efficacy was recently demonstrated under field conditions in a pecan orchard. However, prior to our research, it was unknown whether different soils have differential effects on pheromone enhanced EPN efficacy. In different soils, we tested the biocontrol efficacy of Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora in soil columns with and without pheromone exposure. All nematodes were evaluated in separate columns filled with oven dried commercial play sand and two different soils from pecan orchards (from Byron, GA and Tifton, GA). The soils differed substantially in several aspects such as field capacity, organic matter, nutrients, and nematode movement capacity. Efficacy was determined by baiting the bottom section of each column with larvae of the yellow mealworm (Tenebrio molitor L.). Results indicated that pheromones enhanced EPN efficacy for all EPN species and soils tested compared to treatments without pheromones. The magnitude/extent that pheromones boosted EPN movement in all EPNs regardless of soil type did not differ. Soil did not affect EPN efficacy for H. bacteriophora but did affect S. carpocapsae and S. feltiae. For both S. carpocapsae and S. feltiae efficacy was highest in the sandy field soil (Tifton soil) followed by that of the loamy sand (Byron soil) and pure sand (commercial play sand). When comparing the efficacy of EPN species to each other, we observed that H. bacteriophora killed more bait insects exposed to soil in the bottom of the soil column than other EPNs. Our findings suggest that pheromones can be used to enhance EPN efficacy in diverse soils. Future research may explore pheromone effects on EPNs in additional substrates.

The whitefly, Bemisia tabaci, has a diverse host range that includes many economically important crops. Plant damage and crop loss resulting from infestations of this insect pest averages >$140 million (USD) annually in the southeast U.S. and requires different management approaches. Entomopathogenic nematodes are biological control agents targeting insect pests. The entomopathogenic nematode Steinernema feltiae is particularly virulent against B. tabaci and several other insect pests with aboveground life stages. However, unpredictable efficacy limits the use of entomopathogenic nematodes in aboveground/ foliar pest management strategies. Ascaroside pheromone extracts have been shown to significantly improve the efficacy of entomopathogenic nematodes under field conditions that challenge their survival and efficacy. The objective of this study was to evaluate the influence of ascaroside pheromones on Steinernema feltiae in foliar applications against B. tabaci. Cotton plants were selected as hosts for whiteflies, and 5 ml solutions of nematodes (with and without other treatments) were used for foliar applications. Evaluations occurring through laboratory and cage trials were sampled across seven days. Exposure to the pheromone treatment resulted in higher S. feltiae efficacy as marked by higher B. tabaci adult mortality up to 79.98 % and reduced B. tabaci nymph survival down to 5.92 %. In earlier aboveground trials using entomopathogenic nematodes, Barricade (R) (a gel) facilitated higher efficacy by reducing the risk of desiccation. Barricade (R) was included in foliar applications for comparison and the gel improved entomopathogenic nematodes' efficacy in foliar applications. This study highlights a new avenue for the foliar application of entomopathogenic nematodes with enhanced efficacy.

Plant-parasitic nematodes inflict substantial economic losses on a global scale. While there is a substantial body of knowledge concerning plant-nematode interactions, there is a dearth of information regarding plant-pathogenpathogen interactions when multiple plant-parasitic nematode (PPN) species exist in the vicinity of plants concurrently. In this study, sugar beet plants were employed as a model system to investigate the impact of PPNexposed and non-exposed plant exudates and volatile organic compounds (VOCs) on the orientation behaviors of two economically significant PPN species, root knot nematode (RKN) Meloidogyne incognita and root lesion nematode (RLN) Pratylenchus neglectus. Additionally, the degree to which the exometabolomes (metabolic footprints) of these PPNs influenced nematode behavior, both conspecifically and heterospecifically was measured. Two-choice Petri dish experiments were conducted using root exudates collected from infected and non-infected plants, and the responses of nematodes to these exudates were measured. Similar experiments were conducted using nematode exometabolomes. To test the impact of plant exudates and VOCs on nematode behavior, olfactometer experiments were conducted by placing plants unexposed to nematodes and nematodeexposed plants at the ends of olfactometer arms and nematode responses were assessed after 18-h for RKN and 48-h for RLN. No significant trend was observed in the choices of RLN in the root exudate experiments. In contrast, RKN responded more strongly to exudates from plants previously unexposed to nematodes compared with plants that were exposed to one day and two days post inoculation. No significant differences were found for either nematode species in the exometabolome experiments. In olfactometer trials, RKNs were attracted to uninfected plants over PPN-infected plants or no plant. In the case of RLN olfactometer trials, significant differences were detected, but no specific trend could be established. This study highlights the conspecific and heterospecific behavioral interactions between nematodes exposed to previously infected plant roots.

Disrupting behaviors linked to movement of primary mosquito vectors, such as diel locomotor activity and visual sensitivity, is a novel and plausible malaria control intervention. Diel locomotor activity is an output of arthropod circadian activity and is influenced by factors such as light, temperature, and infection status. The biogenic amines histamine and serotonin (5-HT) are ingested with blood and differ between healthy hosts and those with severe malaria. They regulate malaria parasite infection in Anopheles stephensi, but the degree to which aging, temperature, and infection interact with ingested biogenic amines to influence mosquito behavior was unknown prior to these studies. We provisioned A. stephensi with histamine and 5-HT at healthy- and malaria-associated levels to examine diel locomotor activity of uninfected A. stephensi across lifespan, at temperatures that A. stephensi could encounter within its range, and on Plasmodium yoelii-infected mosquitoes during sporogony. We further evaluated treatment effects on retinal sensitivity of uninfected mosquitoes during light and dark periods typically associated with low and high activity for this crepuscular species. Treatment with malaria-associated levels of histamine and 5-HT significantly increased the locomotor activity of A. stephensi across lifespan and enhanced retinal sensitivity to a broad spectrum of wavelengths at the onset of light. This treatment in combination with higher temperatures also increased activity levels and broadened the peak hours of activity of A. stephensi. Notably, these effects were infection dependent. Together, our data suggest that histaminergic and serotonergic signaling within the gut-brain axis of A. stephensi could be targeted to alter mosquito activity and visual sensitivity as the basis for novel transmission-blocking strategies for malaria control.

Consistent efficacy is required for entomopathogenic nematodes to gain wider adoption as biocontrol agents. Recently, we demonstrated that when exposed to nematode pheromone blends, entomopathogenic nematodes showed increased dispersal, infectivity, and efficacy under laboratory and greenhouse conditions. Prior to this study, the impact of entomopathogenic nematode-pheromone combinations on field efficacy had yet to be studied. Steinernema feltiae is a commercially available entomopathogenic nematode that has been shown to increase mortality in insect pests such as the pecan weevil Curculio caryae. In this study, the pecan weevil was used as a model system to evaluate changes in S. feltiae efficacy when treated with a partially purified ascaroside pheromone blend. Following exposure to the pheromone blend, the efficacy of S. feltiae significantly increased as measured with decreased C. caryae survival despite unfavorable environmental conditions. The results of this study highlight a potential new avenue for using entomopathogenic nematodes in field conditions. With increased efficacy, using entomopathogenic nematodes will reduce reliance on conventional management methods in pecan production, translating into more environmentally acceptable practices.

The tomato (Solanum lycopersicum (Solanaceae)) is particularly susceptible to Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), a pest that directly and profoundly influences tomato yields. Consequently, the early detection of T. absoluta damage intensity on leaves using machine learning or artificial intelligence -based algorithms is crucial for effective pest control. In this ground -breaking study, the galleries generated by T. absoluta were examined via field images using the Decision Trees (DTs) algorithm, a machine learning method. The unique advantage of DTs over other algorithms is their inherent capacity to identify complex and vague shapes without the necessity of feature extraction, providing a more streamlined and effective approach. The DTs algorithm was meticulously trained using pixel values from the leaf images, leading to the classification of pixels within regions with and without galleries on the leaves. Accordingly, the gallery intensity was determined to be 9.09% and 35.77% in the test pictures. The performance of the DTs algorithm, as evidenced by a high precision and an accuracy rate of 0.98 and 0.99 respectively, testifies to its robust predictive and classification abilities. This pioneering study has far-reaching implications for the future of precision agriculture, potentially informing the development of advanced algorithms that can be integrated into autonomous vehicles. The integration of DTs in such applications, due to their unique ability to handle complex and indistinct shapes without the need for feature extraction, sets the stage for a new era of efficient and effective pest control strategies.

Maintenance of an aggregated population structure implies within-species communication. In mixed-species environments, species-specific aggregations may reduce interspecific competition and promote coexistence. We studied whether movement and aggregation behavior of three entomopathogenic nematode species changed when isolated, as compared to mixed-species arenas. Movement and aggregation of Steinernema carpocapsae, S. feltiae and S. glaseri were assessed in sand. Each species demonstrated significant aggregation when alone. Mixed-species trials involved adding two species of nematodes, either combined in the center of the arena or at separate corners. While individual species became less aggregated than in single-species conditions when co-applied in the same location, they became more aggregated when applied in separate corners. This increased aggregation in separate-corner trials occurred even though the nematodes moved just as far when mixed together as they did when alone. These findings suggest that maintenance of multiple species within the same habitat is driven, at least in part, by species-specific signals that promote conspecific aggregation, and when the species are mixed (as occurs in some commercial formulations involving multiple EPN species), these signaling mechanisms are muddled.

Entomopathogenic nematodes (EPNs) are roundworms that parasitize insects with the aid of symbiotic bacteria. These nematodes have been used both as model organisms and for biological control of pests. The specialized third stage of an EPN, known as an infective juvenile (IJ) must forage to find a host with strategies varying from species to species (cruising, ambushing, and intermediate). Some IJs move more than others to find a host, despite an increased risk of predation and desiccation. This hints at potential underlying benefits (e.g., increased invasion) for EPNs that move more. We assessed whether EPNs that moved farther down a soil column also exhibit higher levels of invasion when compared to nematodes that remained at or near their point of origin. We found that movers in the cruisier and intermediate species: Steinernema riobrave, Heterorhabditis bacteriophora, and H. indica had higher invasion rates compared to their counterparts that did not move. S. carpocapsae, an ambusher, did not exhibit invasion differences between EPNs that moved versus those that did not. For the three cruiser/intermediate EPNs we tested, our results support our hypothesis that EPNs that tend to move more enjoy related benefits such as increased invasion potential. Further studies are required to explore other parameters that may interact with movement. The results of this study can potentially be used to develop EPN strains that move more and invade more, and thus can potentially be more effective biological control agents.

Interactions between belowground and aboveground heterotrophic communities with no direct physical contact can be connected by the plant as a mediator. Plants respond to the attack of herbivores by producing a suite of defensive compounds that can affect the choice and performance of other herbivores. The aim of this study was to determine the impact of one herbivore's activity on the acceptability of that plant to another species of herbivore. Two herbivores were tested; root-knot nematode, Meloidogyne incognita (RKN), a belowground plant-parasitic nematode, and the two-spotted spider mite, Tetranychus urticae (TSSM), an aboveground folivore. We conducted herbivore preference and performance tests on Lima bean (LB) (Phaseolus lunatus cv. Henderson) as an optimal host for TSSM, and tomato (Solanum lycopersicum cv. Rutgers) which is a sub-optimal host for TSSM but optimal host for RKN. We used two-choice glass olfactometers to measure the response of RKN to plants that were exposed to TSSM versus a clean LB plant. RKN infected the clean plants at a significantly greater rate than the TSSM exposed plants. TSSM preference was measured, using leaf discs and two-choice olfactometers containing a RKN infected plant versus a clean plant at different days post-inoculation (DPI) of the RKN. TSSM preferred the clean plants to those with 25-day old RKN infections on LB, but preferred RKN infected tomato plants at 1 DPI. We also tested the effect of the inoculation (1 DPI) of the entomopathogenic nematode (EPN) Steinernema carpocapsae on tomato plants and TSSM preferred the EPN inoculated plants. We carried out a non-choice performance test for TSSM on both LB and tomato on plants inoculated with RKN versus clean plants and observed no effect of RKN exposure on TSSM performance. This research shows that plants can mediate interactions between below and aboveground herbivores that share the same plant.

One of the most important development trends of robots in agriculture is to enable highly precise applications that minimize amounts of chemical components that are harmful to the environment. Precision agriculture is fundamental and inevitable worldwide because it provides more yield to an increasing population, while at the same time reducing inputs. The purpose of this study was to apply entomopathogenic nematodes, which are insect parasites used as biological control agents, through Nemabot. A robotic system that can move in the X-Y-Z coordinate plane has an agitating mechanism for suspension based on water and entomopathogenic nematodes and can perform precise dosing with a peristaltic pump designed and produced as a prototype. The experimental results of the robot application on the exact point, volume, amount, and uniformity of dosing show that the proposed method can effectively solve the problem of applying entomopathogenic nematodes, which are economically more expensive than pesticides. The main contribution of this paper is the proposal of a method to solve the problem of applying the agents precisely. This is the first experiment in which biological control products were applied using a robotic system. A patent application (PCT/TR2019/050768) was made, and the patentability claims were approved and officially registered (TR2018 14310B).

Aggregations of foraging animals are key aspects of their ecology, driving spatial patterns, resource access, and successful resource exploitation. Entomopathogenic/insect parasitic nematodes demonstrate aggregated population structures. However, there are gaps in our understanding of how different behaviours affect aggregation. To understand joining behaviour as a mechanism of aggregation, we examined the group movement and joining behaviour of the EPN species, Steinernema glaseri, in conspecific (S. glaseri) and heterospecific (S. carpocapsae and S. feltiae) assemblages. We assessed group movement of S. glaseri using a glass olfactometer where nematodes were added to the central hub and allowed to disperse into six arms towards cues at the ends. We measured movement in the absence of external cues, when host cues were present but uniform, and in response to both con- and heterospecific entomopathogenic nematodes. S. glaseri dispersed in a highly aggregated fashion both in the presence and absence of host cues. When conspecific nematodes were present in the olfactometer ends, S. glaseri readily moved towards and joined conspecific groups, particularly if those conspecifics had experienced host contact 48 h previously. When heterospecific nematodes were present in the ends, S. glaseri only appeared to preferentially join groups of S. feltiae with prior host contact. S. glaseri exhibited no propensity to join groups of S. carpocapsae regardless of prior host contact. Findings demonstrate context-dependent joining behaviours that may underlie aggregation in EPNs. These behaviours may lead to more effective mass attack and regulate interspecific competition among these insect parasites.

Steinernema and Heterorhabditis are not closely related phylogenetically but they share many characteristics via convergent evolution. Over 100 species of Steinernema have been described. Here we focus on Steinernema carpocapsae. All entomopathogenic nematode (EPN) species are symbiotically associated with entomopathogenic bacteria: S. carpocapsae retains Xenorhabdus nematophila. The bacteria kill the insect host along with EPN-produced factors, provide nutrition for the EPNs, and produce metabolites that protect the infection by inhibiting opportunistic colonization by other soil organisms. S. carpocapsae is a common species isolated from soil from every continent except Antarctica and was the first EPN-produced in vitro and commercially developed for pest control. To date, 12 species of EPNs have been commercialized for biological control of soil insect pests for agriculture, municipalities, and homeowners.

Blood levels of histamine and serotonin (5-HT) are altered in human malaria, and, at these levels, we have shown they have broad, independent effects on Anopheles stephensi following ingestion by this invasive mosquito. Given that histamine and 5-HT are ingested together under natural conditions and that histaminergic and serotonergic signaling are networked in other organisms, we examined effects of combinations of these biogenic amines provisioned to A. stephensi at healthy human levels (high 5-HT, low histamine) or levels associated with severe malaria (low 5-HT, high histamine). Treatments were delivered in water (priming) before feeding A. stephensi on Plasmodium yoelii-infected mice or via artificial blood meal. Relative to effects of histamine and 5-HT alone, effects of biogenic amine combinations were complex. Biogenic amine treatments had the greatest impact on the first oviposition cycle, with high histamine moderating low 5-HT effects in combination. In contrast, clutch sizes were similar across combination and individual treatments. While high histamine alone increased uninfected A. stephensi weekly lifetime blood feeding, neither combination altered this tendency relative to controls. The tendency to re-feed 2 weeks after the first blood meal was altered by combination treatments, but this depended on mode of delivery. For blood delivery, malaria-associated treatments yielded higher percentages of fed females relative to healthy-associated treatments, but the converse was true for priming. Female mosquitoes treated with the malaria-associated combination exhibited enhanced flight behavior and object inspection relative to controls and healthy combination treatment. Mosquitoes primed with the malaria-associated combination exhibited higher mean oocysts and sporozoite infection prevalence relative to the healthy combination, with high histamine having a dominant effect on these patterns. Compared with uninfected A. stephensi, the tendency of infected mosquitoes to take a second blood meal revealed an interaction of biogenic amines with infection. We used a mathematical model to project the impacts of different levels of biogenic amines and associated changes on outbreaks in human populations. While not all outbreak parameters were impacted the same, the sum of effects suggests that histamine and 5-HT alter the likelihood of transmission by mosquitoes that feed on hosts with symptomatic malaria versus a healthy host.