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For delaying evolution of pest resistance to transgenic corn producing Bacillus thuringiensis (Bt) toxins, limited data are available to compare the effectiveness of refuges of non-Bt corn planted in seed mixtures versus blocks. Here we addressed this issue in the ear-feeding pest Helicoverpa zea Boddie by measuring its survival and development in the laboratory on ears from field plots with 90% Cry1A.105 + Cry2Ab corn and 10% non-Bt corn planted in a seed mixture or blocks. We compared a strain of H. zea selected for resistance to Cry1Ac in the laboratory, its parent strain not selected in the laboratory, and their F1 progeny. The relative survival of the F1 progeny and dominance of resistance were higher on ears from Bt plants in the seed mixture than the block. Half of the kernels in ears from non-Bt plants in the seed mixture produced both Cry1A.105 and Cry2Ab. However, survival on ears from non-Bt plants did not differ between the block and seed mixture. In simulations based on the observed survival, resistance to Cry1A.105 + Cry2Ab corn evolved faster with the seed mixture than the blocks, because of the higher dominance of resistance in the seed mixture. Increasing the refuge percentage improved durability of Cry1A.105 + Cry2Ab corn more for the blocks than the seed mixture. These findings imply that, for a given percentage of non-Bt corn, resistance of H. zea and other ear-feeding pests to multi-toxin Bt corn is likely to evolve faster for seed mixtures than blocks.
During a trapping study interval, each target insect is either caught or not caught. Therefore, the current analysis treats trapping as a binomial process. Data from a binomial calculator, along with computer simulations of random walkers, documented that the inherent variance associated with estimates of absolute population density generated by a single catch number in a pheromone-baited monitoring trap becomes very high when catch probability averaged across the trap's sampling area falls below 0.02, as is the case for most insect trapping systems operating in the open field. The imprecision associated with interpretations of single catch numbers renders many current pest management decisions risky and unsatisfactory. Here we reinforce how single-trap, multiple-release experiments can and should be used to measure catch probability, plume reach, and trap sampling area. When catch probability lies in the danger zone below 0.02, steps are suggested for how multiple traps might be deployed to raise composite catch probability to a level where estimates of absolute pest density become reliable. Heat transfer is offered as an appropriate conceptual model for the mechanics of trapping. A call is made for a radical rethinking in the designs of insect monitoring traps in light of their significant current deficits highlighted by this study.
Here, we review the literature on the development and application of hydrogel compounds for insect pest management. Researchers have used hydrogel compounds for the past few decades to achieve the controlled release of various contact insecticides, but in recent years, hydrogel compounds have also been used to absorb and deliver targeted concentrations of toxicants within a liquid bait to manage insect pests. The highly absorbent hydrogel acts as a controlled-release formulation that keeps the liquid bait available and palatable to the target pests. This review discusses the use of various types of hydrogel compounds in pest management based on different environmental settings (e.g., agricultural, urban, and natural areas), pest systems (e.g., different taxa), and modes of insecticide delivery (e.g., spray vs bait). Due to their unique physicochemical properties, hydrogel compounds have great potential to be developed into new and efficacious pest management strategies with minimal environmental impact. We will also discuss the future research and development of hydrogels in this review.
Nosema disease is a prominent malady among adult honey bees [Apis mellifera L. (Hymenoptera: Apidae)], caused by the microsporidian parasites, Nosema apis Zander (Microspora: Nosematidae) and N. ceranaeFries et al. 1996.The biology of N. apis is well understood, as this parasite was first described over a century ago. As N. ceranae is an emerging parasite of the honey bee, we do not yet understand how long spores of this parasite survive in honey bee colonies, or all the potential modes of transmission among bees. We investigated the viability and infectivity of N. ceranae spores in honey and on beeswax over time after exposure to 33, 20, –12, and –20°C. Spores in honey maintained viability at freezing temperatures for up to 1 yr and remained viable considerably longer than those on beeswax. Based on this evidence, honey may act as an important reservoir for infective spores to initiate or perpetuate N. ceranae infections in honey bee colonies. This work provides information that may help enhance current management recommendations for apiculturalists.
Our previous study provided correlative evidence that morning glory species harboring endophytic fungi (Periglandula) are resistant to potato psyllid [Bactericera cockerelli (Šulc)], whereas species free of fungi often allowed psyllid development. In this study, we manipulated levels of ergot alkaloids in host tissues by inoculating clippings from potato plants with extracts from morning glories that harbor Periglandula [Ipomoea leptophylla Torrey, Ipomoea imperati (Vahl) Grisebach, Ipomoea tricolor Cavanilles, Ipomoea pandurata (L.) G. F. Meyer, and Turbina corymbosa (L.)] and one species (Ipomoea alba L.) that does not harbor the endophyte. Ergot alkaloids (clavines, lysergic acid amides, and ergopeptines) were detected in potato clippings, thus confirming that leaves had taken up compounds from solutions of crude extracts. Psyllid mortality rates on inoculated clippings ranged between 53 and 93% in treatments producing biochemically detectable levels of alkaloids, when compared with 15% mortality in water controls or the alkaloid-free I. alba. We then tested synthetic analogs from each of the three alkaloid classes that had been detected in the crude extracts. Each compound was assayed by inoculating clippings of two host species (potato and tomato) at increasing concentrations (0, 1, 10, and 100 µg/ml in solution). Psyllids exhibited a large and significant increase in mortality rate beginning at the lowest two concentrations, indicating that even very small quantities of these chemicals led to mortality. Feeding by nymphs on artificial diets containing synthetic compounds resulted in 100% mortality within 48 h, irrespective of compound. Further testing of ergot alkaloids to characterize the mode of action that leads to psyllid mortality is warranted.
Helicoverpa armigera Nucleopolyhedrovirus (HearNPV) (genus: Alphabaculovirus, incertae sedis: Baculoviridae) has been used to control Helicoverpa armigera (Hübner). A reproducible and susceptible cell line was prepared from the hemocytes of Ephestia kuehniella in Grace and Ex-Cell 420 media. The population doubling time of these cloned cell cultures during the logarithmic phase were about 2.3 and 3.7 d for Ex-Cell 420 and Grace's media, respectively. When 60% confluence occurred, cells were infected by viral inoculums. All biochemical compounds were significantly changed relevant to cellular metabolism due to HearNPV infection. In order to improve its stability, two polymer formulations were used, i.e., formulation A (sodium alginate, gelatin, starch, and molasses) and formulation B (cottonseed kernel extract, Bran, glycerol, boric acid, egg white, and sugar). Formulant A provided high photostability by exhibiting 83.2 ± 3% efficacy and 88.66 ± 2.1% original activities remaining after 72 h UV exposure. Percentage original activity remaining of unformulated HearNPV and formulated mixture of B was 38.66 ± 2.6% and 9.33 ± 1.3%, respectively, after 72 h UV-irradiation. The virulence of the HearNPV proliferated from the Ex-Cell medium was similar to the virulence of wild-type HearNPV with LC50 of 7.7×105 OBs/ml. Formulant A, revealed only 20.0 ± 1% reduction in efficacy while the unformulated virus and formulant B faced a reduction of 90.0 ± 3% and 64.0 ± 2% after 72 h of UVA irradiation. Formulant A thus showed a high potential to protect HearNPVs microparticles against UV-inactivation suggesting a new platform for more efficient biological-management of cotton bollworm (specific name Helicoverpa armigera, genus: Helicoverpa, Lepidoptera: Noctuidae) in vivo.
The twospotted spider mite (TSSM, Tetranychus urticae Koch) is a key pest of tomatoes (Solanum lycopersicon L. [Solanales: Solanaceae]) in North Carolina, and its management has relied principally on synthetic acaricides. Augmentative biological control of TSSM is a commonplace and effective management strategy in greenhouses worldwide, but in field-grown vegetable crops biocontrol of TSSM is poorly developed. We conducted small-plot field experiments in 2016 and 2019 to test the ability of the predatory mite, Phytoseiulus persimilis Athias-Henriot, to establish, disperse, and reduce TSSM in staked tomatoes, as well as test their compatibility with a commonly used reduced-risk acaricide, bifenazate (Acramite). Across both years, the most effective treatment for reducing TSSM was the combination of P. persimilis + Acramite. Phytoseiulus persimilis successfully established both years, but its population growth and dispersal were greater in 2016 than 2019. Acramite alone significantly reduced TSSM pressure in 2019 and P. persimilis alone did not reduce TSSM pressure below the control in either year. However, results in 2016 were influenced by the eventual dispersal of P. persimilis into all experimental plots, despite efforts to hinder their movement with corn barriers. Results in 2019 were influenced by the late-season invasion of TSSM into the trial, which delayed P. persimilis releases and influenced their establishment and growth. This study is the first to demonstrate the utility and limitations of P. persimilis in staked field tomatoes, which is a promising option for growers to manage TSSM in tomatoes in the southeast United States.
Wireworms are destructive soil inhabiting polyphagous pests in the Pacific Northwest and Intermountain region of the United States. Continuously increasing wireworm populations and damage in small grain crops such as spring wheat in Northern Great Plains has become a challenge for growers. Due to unavailability of effective control measures, alternative methods, including biological control agents such as entomopathogenic nematodes (EPNs) are needed. Native/indigenous EPN species are expected to have better potential than exotic species to control the local insect pests. Two Montana native EPN species (Steinernema feltiae and Heterorhabditis bacteriophora) were tested against, Limonius californicus (Coleoptera: Elateridae) in laboratory and shade house studies. In the laboratory bioassay, two isolates of S. feltiae at the rate of 28,000 IJs/ five larvae killed 48–50% of the insects within 4 wk. Heterorhabditis bacteriophora was not able to cause >30% L. californicus larval mortality. None of the two isolates of S. feltiae performed well against L. californicus when tested in different soil types. Similarly, two isolates of S. feltiae that were tested killed only 20–25% wireworms in a shade house trial that did not differ significantly from the control treatment. Four weeks after EPN treatment in the shade house trial, the percentage of wheat plant damage from L. californicus ranged from 30 to 40% in the presence of S. felitae, not differing statistically from control. These results suggest that S. felitae have limited potential in managing wireworm populations.
Ontsiramellipes Ashmead is a gregarious larval ectoparasitoid of woodboring cerambycids that is native to North America but can readily attack the exotic Asian longhorned beetle, Anoplophora glabripennis (Motschulsky). To evaluate the potential of the parasitoid as a novel association control agent for the pest beetle, this study investigated some key reproductive traits of the parasitoid, including egg maturation dynamics, and host size preference and suitability in association with the beetle. Results showed that female wasps emerged with a substantial portion (38%) of their lifetime complement of mature eggs and matured eggs rapidly, reaching a peak 4–6 d post-eclosion. The number of mature eggs was positively related to the female wasp's body size. Oviposition prompted production of more mature eggs by young female wasps. The parasitoid did not show a significant preference for large over small hosts in a choice test. Host size did not affect the parasitoid's offspring survival, developmental time, or sex ratio. However, clutch size increased with increasing host size. Female wasps that developed from large hosts had larger body size and consequently a higher mature egg load than those reared from small hosts. Neither longevity nor the total number of parasitized hosts over a female's lifetime was affected by the female's size, but the total number of offspring produced per female increased with the female's size. These results have important implications for improving rearing and field-release strategies as well as understanding the ecological mechanisms underlying host size selection in gregarious parasitoids.
Euwallacea fornicatus (Eichhoff), the polyphagous shot hole borer (PSHB), is an ambrosia beetle infesting avocado Persea americana Mill. limbs in North America and Israel. We conducted field experiments with sticky traps in avocado orchards to develop push–pull semiochemical methods of managing PSHB. Traps baited with 10-fold increasing doses (0.01 to 100× or 1.26 µg to 12.6 mg/d) of attractant quercivorol were previously shown to increasingly capture female PSHB (males flightless). We converted trap catch of this relationship to a standardized effective attraction radius (EAR) that predicts capture power of baited-traps regardless of insect flight density. Earlier, piperitone and verbenone were shown to strongly inhibit attraction of PSHB to quercivorol-traps. We tested increasing numbers of 1× piperitone dispensers at 0.75-m distance surrounding a quercivorol-trap and found PSHB catch to decline exponentially. Increasing decadic doses (0.01 to 10×) of either verbenone or piperitone released at 1× quercivorol-traps caused a sigmoidal first-order kinetic-decay in catch. Verbenone (1×) placed at increasing distances (0, 0.25, 0.5, 1, and 2 m) from a 1× quercivorol-trap became increasingly ineffective in reducing catch of PSHB. We found no evidence that ethanol released from 7.5 to 480 mg/d affected attraction of PSHB, but Scobicia chevrieri (Villa and Villa) (Coleoptera: Bostrichidae) was increasingly attracted. Due to their relatively short-range (<0.5 m) inhibition of attractive sources, piperitone, and verbenone dispensers should be placed on avocado trunks where PSHB aggregations occur before the flight season.
The black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae), is mass produced worldwide for use in waste management and the production of an alternative protein for use as feed. However, few publications have explored its adult behavior, particularly mating, as a means to optimize egg production in colony. In addition, there is limited knowledge of appropriate marking techniques to explore the mating behavior of this insect.The goal of this study was to validate water-based acrylic paint pens as a possible marking tool for behavioral studies with adult black soldier flies. Adult black soldier flies (<24-h old) were marked with green, gold, red, or white acrylic paint. Adult longevity, as well as the number of matings (mating success) were recorded for each treatment. Regardless of marking, 1) adult females lived ∼7 d, whereas males lived 9 d, 2) mating frequency peaked 2 d after initiation of the experiment, 3) approximately 30% of adults across treatments were recorded mating, and 4) egg production and hatch rate were not affected.This marking technique did not affect the parameters measured, suggesting that it is suitable for experiments requiring a reliable marking technique.
Many lepidopteran species rely upon active flight to migrate over long distances, thus pursuing ephemeral resources, colonizing new habitats, or escaping adverse meteorological conditions. Though their biology and ecology are often well studied, there is only scant information on their wingbeat frequency (WBF), a key aerodynamic determinant of insect flight. In this study, we assessed interspecific and seasonal variability in WBF for 85 different migratory species of Lepidoptera (11 families) under the laboratory conditions of 25 ± 1°C and 75 ± 5% RH. WBF of migrant individuals ranged between 6.7 and 84.5 Hz and substantial interspecific differences were recorded, with members of the Bombycidae exhibiting the highest mean WBFs (i.e., 55.1 ± 1.0 Hz) and Saturniidae the lowest (8.5 ± 0.2 Hz). At a species level, seasonal variation was observed in WBF for Mythimna separata (Walker) (Lepidoptera: Noctuidae), Scotogramma trifolii Rottemberg (Lepidoptera: Noctuidae), and Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). Our findings add to the scientific knowledge on flight biology of migratory insects, facilitate (automatic) monitoring and population forecasting, and can have broader implications for insect pest management or biodiversity conservation.
The fruit flies Bactrocera tau (Walker) and B. cucurbitae (Coquillett) are economically important invasive pests on numerous vegetable and fruit species in China. Due to the instability of the early spring climate, temperatures often deviate far below the normal temperature for short periods of time. Such a sudden short-term low temperature may impact the reproduction and development of the two fruit fly species. In this study, the effects of low temperatures (8, 6, 4, 2, 0, –2, and –4°C) on the development and reproduction of these two closely related fruit fly species were studied under laboratory condition. The results showed that their survival rates decreased gradually with corresponding decreases in the tested temperatures. On the other hand, their pre-oviposition periods and their adult female to male sex ratios increased, while the average number of eggs per female of each species and longevity of male adult of B. cucurbitae initially increased and later decreased after exposure to the low-temperature treatments. Overall, low temperatures promoted reproduction in B. cucurbitae and B. tau at temperatures ranging from 24°C to as low as 8°C. Development and reproduction of the two species were negatively affected when temperatures were between 8 to –4°C. The cold resistance of each developmental stage was higher in B. tau than in corresponding stages of B. cucurbitae.
There is a paucity of information on the edible grasshoppers and their host plants in East Africa. This study adopted morphological and molecular analysis to identify edible grasshoppers in Kenya and Uganda. The associated host plants were identified through molecular analysis of the gut contents of the grasshoppers. The cytochrome b and 16s gene primers were used for grasshopper DNA analysis; while matK gene primers were used for plant DNA analysis. All long-horned grasshoppers sampled were identified as Ruspolia differens (Serville) (Orthoptera: Tettigonidae); whereas short-horned grasshoppers were identified as Acanthacris ruficornis (Fabricius) (Orthoptera: Acrididae) and Cyrtacanthacris tatarica (L.) (Orthoptera: Acrididae). Host plants of A. ruficornis were Achyranthes aspera (L.), Centella virgata L.f. Drude, Digitaria gayana (Kunth), Galinsoga quadriradiata Ruiz and Pavon, and Triumfetta pilosa Roth; whereas those of C. tatarica were Alysicarpus rugosus (Willd.) DC and Teramnus uncinatus (L.) SW. Host plants of R. differens were Ageratum conyzoides (L.), Citrus depressa Hayata, Cynodon dactylon (L.), D. gayana, Eragrostis mexicana Hornem, Eucalyptus saligna SM., Indigofera arrecta Hochst. ex A. Rich., Persicaria nepalensis (L.), and Sorghum halepense (L.). Information on the host plants of edible grasshoppers can help in the development of their mass rearing protocols.
Anastrepha ludens (Loew) (Diptera: Tephritidae) is an endemic pest of Mexico, attacking several fruits of economic importance. The Sterile Insect Technique (SIT), consisting of mass-rearing, irradiation and release of adults, is used to control this pest in affected areas. Currently, a genetic sexing strain (Tapachula 7, Tap-7) consisting only of A. ludens males is being released, yet we lack information on its sexual performance at the early ages when they are released and on its post copulatory behavior in terms of sperm transfer and mating inhibition. Here, sexual competitiveness at young ages and ability to inhibit female re-mating was compared between sterile Tap-7, Standard Bisexual (SB), or wild males both in laboratory and field cage conditions. Sperm stored by females mating with wild, Tap-7, or SB strain males was also compared. Six-day-old sterile Tap-7 males had low mating rates, however, by 7 d of age 80% of males had mated. Tap-7 males were just as likely as wild males to inhibit wild female re-mating. In field cages, sterile Tap-7 males mated faster than wild and sterile males from the SB strain and had comparable mating success to wild males. Females mated to sterile Tap-7 males stored more sperm than those mated to sterile SB males. Females mated to wild males stored more sperm than females mated to any other type of male. Mass-rearing had a greater effect on decreasing sperm stored by females than irradiation. We recommend continuing the release of the GSS strain of A. ludens.
Cockroaches are important sanitary pests and very difficult to control worldwide. With public concern about traditional insecticides, cockroach control agents should be environmentally friendly, highly efficient, and economical. In this article, 12 essential oils were screened to test their repellent effect against Blattella germanica. To develop essential oils as repellent agents, the oils were further examined in binary synergistic combinations. Ilex chinensis Sims (Sapindales: Aquifoliaceae) oil, Lavandula spp (Tubiflorae: Labiatae) oil, and Elsholtzia ciliata (Thunb.) Hyland (Tubiflorae: Labiatae) oil showed excellent repellent activities with lower RD50 (repellency dose for 50% of treated adults) values of 218.634, 154.590, and 223.989 µg/cm2, respectively, compared to those of other oils and the positive control. The I. chinensis oil and E. ciliata oil (weight ratio of 1:1.41) combination also displayed a remarkable synergistic effect against B. germanica. Their cotoxicity coefficient was 214.4. The major chemical constituents in E. ciliata and I. chinensis oils were respectively 3,7-dimethyl-1, 6-octadien-3-ol and methyl salicylate. The binary oil mixtures were formulated as a sustained release agent with γ-CD. The optimal preparation should be an 8:1 ratio of γ-CD to oils, with a 1 h stirring time, 50°C stirring temperature, and 1:12 ratio of γ-CD to ddH2O. The results of this study suggest that sustained release of binary oil-γ-CD exhibited a prolonged repellent activity (10 h) against B. germanica. This sustained-release agent could be further investigated and developed as a novel repellent preparation.
Biological stimulation induced by low doses of toxicants or other stressors is known as hormesis. Hormetic stimulation of life history traits in insect pests can negatively impact agriculture, but stimulation of beneficial insects could be leveraged to enhance biological control agents. We examined whether low doses of imidacloprid could enhance oviposition, fecundity, fertility, and survival in the beneficial stink bug predator, Podisus maculiventris (Say) (Hemiptera: Pentatomidae), exposed at different life stages and across two generations. When treated as young adults, P. maculiventris fecundity was stimulated at 0.5 and 1.0 mg/liter imidacloprid (<2% of the field rate) without changes in time to oviposition, fertility, and survival. Nymphs exposed to 0.015 mg/liter imidacloprid (<1% of the field rate) also had stimulated reproduction without effects on oviposition, fertility, and survival, but treatment of nymphs at 0.15 and 1.5 mg/liter imidacloprid stimulated fecundity at the expense of fertility and survival. In another experiment we found reproductive stimulation can occur trans-generationally without major reduction in fertility or survival. Our results suggest biocontrol producers may be able to strategically apply low doses of stress to natural enemies during culturing without compromising fitness in subsequent generations.
Fusarium graminearum Schwabe (Hypocreales: Nectriaceae) and Fusarium verticillioides (Saccardo) (Hypocreales: Nectriaceae) Nirenberg infection results in accumulation of deoxynivalenol (DON), zearalenone (ZON), and fumonisin (FBs) mycotoxins in infected corn, Zea mays L. Lepidopteran insect feeding may exacerbate fungal infection by providing entry points on the ear resulting in increased mycotoxin contamination of grain. The objective of the current study was to simulate different types and severity levels (extent of injury) of lepidopteran injury to corn ears at different stages of ear development and its effect on mycotoxin accumulation in grain corn. Field experiments were conducted under conditions favorable for F. graminearum development where insect injury was simulated to corn ears and inoculated with F. graminearum. All simulated injury treatments resulted in elevated mycotoxin concentration compared with ears without simulated injury; however, the severity of injury within a treatment had little effect. Injury to kernels on the side of the ear resulted in greater DON and ZON concentration than injury to tip kernels, grazing injury applied at physiological maturity, or when no injury was simulated. Greater FBs was measured when tip kernel injury was simulated at the blister stage or when side kernel injury was simulated at milk and dent stages compared with noninjured ears, silk clipping, tip injury at milk and dent stages, or grazing injury at physiological maturity. The current study confirms that the risk of mycotoxin accumulation in the Great Lakes region is greater in the presence of ear-feeding insect pests and may differ depending on the feeding behavior of pest species.
A 4-yr study was conducted comparing the efficacy and value of fungicide-only (FST), neonicotinoid insecticide + fungicide (NST), and diamide insecticide + fungicide (DST) seed treatments for commercial corn Zea mays L. and soybean Glycines max (L.) Merr. production in Ontario, Canada. Plant stand, plant vigor, above- and below-ground insect injury, and yield were assessed on 160 field-scale experiments. Experiments also assessed early-season insect incidence and abundance using newly legislated thresholds for NST use in Ontario and in-season destructive sampling.Wireworms (Coleoptera: Elateridae) and white grubs (Coleoptera: Scarabeidae) were frequently observed at experimental sites; however, thresholds were rarely met and injury levels rarely led to yield loss. Of 129 and 31 corn and soybean sites, 8 and 6%, respectively, had a positive yield response to NST use. Across all sites, yield response of 0.1 and –0.05 Mg ha–1 was observed with NST use in corn and soybean, respectively; however, the costs associated with NST use were recovered at only 48 and 23% of corn and soybean sites, respectively, based on average grain prices and yields during the study. Infrequent incidence of economic injury and the absence of a consistent yield response to NST and DSTs throughout the 4 yr of the study indicate that widespread use of seed-applied insecticides in corn and soybean is unlikely to provide benefit to producers.These data highlight an opportunity for reducing input costs, environmental loading, and nontarget effects without adverse outcomes for Ontario producers.
In the United States, the average field size has roughly doubled from the 1980s to the mid-2000s, while average cropland has stayed the same. This will likely influence how semi-natural habitats and edges affect local patterns and processes such as natural pest control or pest densities.We hypothesized that densities of two cotton pests, cotton fleahopper (Pseudatomoscelis seriatus) and verde plant bug (Creontiades signatus) (Hemiptera: Miridae), and corresponding cotton injury in a cotton agroecosystem were affected by field edge, ecotone type (described by the neighboring habitat), and the influence of ecotone type on edge effects. Studies over 2 yr using transect and random point sampling indicated that densities of both insects declined significantly and in a linear fashion from the cotton field boundary (0 m) to field interior (200–300 m from field edge). The decline was influenced by ecotone type for cotton fleahopper. Pest densities in cotton at the interface with semi-natural habitat were higher but declined at a greater rate into the cotton field interior compared to densities seen at the interfaces with sorghum or another cotton field. These effects were also observed for verde plant bug and the cotton boll injury it causes. Regardless of the pest densities near the field edge and the rate of decline into the field interior, densities beyond 100 m into the field were up to 70% less than field edges for both insect species and for boll injury. Potential for land managers to improve sampling efficiency when scouting is apparent. For example, pest species may be at economic threshold in certain parts of the field but not others, leading to different management decisions in larger fields. Therefore, for cotton fleahopper and verde plant bug, edges should be the focus of initial pest detection and sampling, and interior field sampling may only be required when edges are above the economic threshold.
Economically damaging infestations of Lygus lineolaris (Palisot de Beauvois), tarnished plant bug, were sporadic in Virginia and North Carolina cotton before 2010. Given the sudden rise of L. lineolaris as an economic pest in these states, regionally specific management practices (i.e., chemical and cultural control) are needed to help producers protect yield potential while minimizing input costs. Field experiments were conducted in Virginia and North Carolina in 2018 and 2019 to 1) determine the effects of various management practices on L. lineolaris density, plant injury (i.e., square retention, internal boll injury), and lint yield, 2) calculate the economic trade-offs between integrated pest management (IPM) systems approach and current management practices for L. lineolaris in these states, and 3) evaluate economic benefits associated with various sampling thresholds. Lygus lineolaris populations peaked mid-season (i.e., August) during cotton flowering in both states. Weekly scouting and applying foliar insecticides when the current University Extension recommended economic threshold was reached was the most critical management treatment in maximizing economic returns. Additional costs among various IPM practices did not translate into significant yield protection and economic gains. Moreover, there were additional economic benefits associated with protecting glabrous and longer maturing varieties in Virginia. Lygus lineolaris density varied significantly between states; therefore, management recommendations should be modified based on the growing region. Results from this study will be used to create an IPM strategy to help cotton producers effectively manage this insect pest in the Southeast.
An experiment was conducted at the Delta Research and Extension Center in Stoneville, MS during 2017 and 2018 to determine whether removal of the flood is an economical method of control for rice water weevil, Lissorhoptrus oryzophilus Kuschel. This experiment compared a continuous flood production system to draining a rice field completely and reestablishing a flood for the remainder of the growing season. In addition, two insecticide seed treatments, thiamethoxam and chlorantraniliprole, were compared with an untreated control within each system. Rice water weevil densities were measured prior to draining at 3 wk after flood and again after the flood was reestablished in drained plots. Rice water weevil densities were greater in 2017 than 2018. Chlorantraniliprole at the predrainage and postdrainage sample timing reduced larval numbers compared with the untreated control. The plots where water was removed until soil cracking then re-flooded had significantly lower weevil populations than plots that were continuously flooded during 2018 only. Draining of plots resulted in lower yields in 2018, but not in 2017. Additionally, both of the insecticide seed treatments resulted in greater yields and economic returns than the untreated control. Draining of flooded rice when rice water weevil larvae were present did not provide a consistent benefit, and may result in yield and economic penalties. Insecticide seed treatments consistently provided greater yield benefits in flooded rice. Based on these results, draining of flooded rice is not recommended to manage rice water weevil and insecticide seed treatments should be used to minimize economic losses.
Economic yield loss and reduction in grain quality from brown stink bug, Euschistus servus (Say), feeding injury in early and late stages of maize, Zea mays (Poales: Poaceae, Linnaeus), development was assessed in Virginia and North Carolina in 2018 and 2019. Varying levels of stink bug infestations were introduced to seedling maize (V2—early stage), and a range of late-stages of maize, including 1) the last stage of vegetative development (V12/V14), 2) prior to tasseling, 3) at tasseling (VT), and 4) across all tested late growth stages. Euschistus servus infestation levels included 33, 67, and 100% of maize seedlings, and 25, 50, 100, and 200% of plants during later stages. Infestations were maintained on seedling maize for 7 d, and 8 or 16 d in reproductive stages. Infestation level in seedling maize had an impact on grain yield. Infestation level and growth stage both had an impact on grain yield in reproductive maize. The percentage of discolored kernels was also affected by infestation level, but not growth stage. Regression analysis between grain yield and infestation level indicated that the average economic injury level is 7% in seedling maize (7 bugs/100 plants) and 12% (12 bugs/100 plants) from the last vegetative stages (V12/V14) through pollination (VT). The economic injury level in the late vegetative stages is only applicable when infestations are present for an extended period of time (16 d), emphasizing the need for continued scouting of maize throughout the season to make informed management decisions.
Bacillus thuringiensis Cry proteins have been widely used over the past decades for many different insect pests, which are safe for users and the environment. The coleopteran-specific Cry3Aa toxin from B. thuringiensis exhibits toxicity to the larvae of Monochamus alternatus. Receptors play a key role in the mechanisms underlying the toxic action of Cry. However, the binding receptor for Cry3Aa has yet to be identified in the midgut of M. alternatus larvae. Therefore, the aim of this study was to identify the receptor for Cry3Aa toxin in the brush border membrane vesicles (BBMVs) of M. alternatus larvae. Our results indicate that the Cry3Aa toxin binds to the BBMVs (Kd = 247 nM) of M. alternatus via a 107 kDa aminopeptidase N (APN) (Kd = 57 nM). In silico analysis of the APN protein predicted that an 18 amino acid sequence in the N-terminal acted as a signal peptide, and that the Asn residue, located at position 918 in the C-terminus is an anchored site for glycosyl phosphatidyl inositol. Further analysis showed that M. alternatus APN exhibits 75% homology to the APN from Anoplophora glabripenis. Our work, therefore, confirmed that APN, which is localized in the BBMVs in the midgut of M. alternatus larvae, acts as a binding protein for Cry3Aa toxins.
The wood-boring larvae of longhorned beetles (Coleoptera: Cerambycidae) can be important pests of woody plants, particularly as invasive species introduced by international commerce. Previous research has revealed that cerambycid species native to different parts of the world often share the same aggregation-sex pheromones and that pheromones of different species can be combined to create multi-species attractants that would be advantageous for surveillance monitoring for a number of species simultaneously. To explore the extent to which these chemicals can be combined into single lures, we developed four different blends of six to eight compounds and tested their effects as attractants for a community of longhorned beetle species in Iowa. The blends included known pheromones of species native to the study site, as well as pheromones identified from cerambycid species native to other parts of the world. The experiment confirmed that several cerambycid species were attracted by specific blends, in accordance with their known pheromone chemistry, and despite the presence of pheromone components of heterospecifics. This finding lends further support to developing multi-component blends that can effectively monitor for new incursions of multiple exotic species concurrently.
The cycad aulacaspis scale, Aulacaspis yasumatsui Takagi (Hemiptera: Coccoidea: Diaspididae), is native to Southeast Asia but an invasive pest of the gymnosperm order Cycadales in many parts of the world. Aulacaspis yasumatsui was recently reported on the cycad genus Encephalartos in South Africa and is currently categorized as a ‘prohibited terrestrial invertebrate’ in the invasive species legislation, National Environmental Management: Biodiversity Act, 2004 (NEM:BA). Encephalartos is endemic to Africa, and 11 species are listed as critically endangered and four species as endangered. Seeing the limited distribution of A. yasumatsui in South Africa and only one unconfirmed record from the Ivory Coast, understanding the potential distribution range is essential for control and management. Here we model the potential distribution of A. yasumatsui under current and future climate scenarios in Africa, with a focus on South Africa. Future climatic scenarios were simulated using a bio-climatic software, CLIMEX. The model indicates that, under the current climatic scenario, all 17 African countries possessing Encephalartos are susceptible to A. yasumatsui establishment. However, under climatic change, the suitability decreases for large parts of Africa. In South Africa, 93% of the winter rainfall areas, and 90% of the temperate, summer rainfall areas are suitable for A. yasumatsui establishment. In this study, we highlight the urgent need for regulation, management, and research on A. yasumatsui in African countries with native cycads.
Three ‘Red Delicious,' Malus domestica Borkhausen (Rosales: Rosaceae), apple plantings, each representing a different sport, were evaluated for natural resistance against the obliquebanded leafroller (OBLR), Choristoneura rosaceana (Harris). The establishment of neonate larvae on apple foliage was not different between the three ‘Red Delicious' plantings. Of the three ‘Red Delicious' plantings, the one that most negatively impacted OBLR was the ‘It Delicious' genotype. The ‘It Delicious' genotype at the Sunrise Research Orchard exhibited essentially 100% mortality against OBLR when fed on spring and summer foliage, and mortality accumulated faster across instars than on other ‘Red Delicious' plantings. The high mortality observed in the ‘It Delicious' genotype points to the existence of a putative gene, which we propose as Cro1. The other ‘Red Delicious' plantings, Columbia River Orchard and Tree Fruit Research and Extension Center Research Orchard treatments, showed negative impacts, especially when exposed to foliage from the summer compared to the spring period. Development rates in these treatments in spring were higher compared to summer, and there were direct relationships between development rates, pupal weights, and adult longevity for both males and females. These latter results suggest that sublethal effects could be present in these ‘Red Delicious’ cultivars, thus offering insights to a gene-pyramiding strategy for breeders to managing leafroller pests in Washington apple.
Erythritol is a dietary sweetener that is used for low-calorie or diabetic diets. Although safe for human consumption, erythritol is lethal to certain Dipteran pests, but insecticidal effects of erythritol on phloem-feeding insects have yet to be examined. Our goal was to determine whether erythritol has insecticidal activity against pear psylla, Cacopsylla pyricola (Foerster) (Hemiptera: Psyllidae). We first demonstrated that ingestion of erythritol solutions compared with water by pear psylla caused reduced feeding, impaired motor functions, and reduced survival time of adults. We then tested whether foliar treatment of pear leaves with erythritol was also lethal to pear psylla. Foliar treatment of erythritol led to reduced 3-d survival of pear psylla nymphs and adults, and reduced rates of oviposition by pear psylla adults. Psylla adults also preferred to settle on untreated leaves than on erythritol-treated leaves in preference assays. Finally, we conducted field experiments to test whether applications of erythritol provided pear trees with protection against pear psylla under natural field conditions. Those experiments showed a reduction in pear psylla nymphs on erythritol-treated trees compared with untreated trees, but only if the erythritol was completely dissolved into solution by heating. Laboratory trials confirmed the importance of heating. Results of our experiments demonstrate that erythritol is insecticidal to pear psylla nymphs and adults and provide the first report that erythritol is lethal to a phloem-feeding insect. These findings suggest that erythritol may provide a new safe and effective tool for the management of pear psylla.
Allium leafminer, Phytomyza gymnostoma Loew, is the newest invasive pest of allium crops in North America. Larvae initially feed in the upper canopy before mining toward the base of the plant to pupate. Crop loss occurs when larvae destroy vascular tissue, facilitating infection by bacterial and fungal pathogens that cause rot. Contamination also occurs when larvae and pupae are present at harvest. In response to this invasion, efficacy of 14 insecticide active ingredients applied via foliar sprays, transplant treatments, and drip chemigation was evaluated for managing P. gymnostoma. Multiple field studies were conducted in onions, leeks, and scallions in Pennsylvania and New York, United States in 2018 and 2019. The highest and most consistent levels of P. gymnostoma control occurred using foliar applications of dinotefuran, cyantraniliprole and spinetoram (84–89% reduction in damage; 95% reduction in P. gymnostoma densities). Despite the success of dinotefuran and cyantraniliprole applied as foliar sprays, neither was effective in controlling P. gymnostoma when administered via drip chemigation. Other foliar-applied insecticides that significantly reduced densities of P. gymnostoma in one or two experiments included abamectin, acetamiprid, cyromazine, imidacloprid, lambda-cyhalothrin, methomyl, and spinosad. Active ingredients that never controlled P. gymnostoma included azadirachtin, kaolin clay, pyrethrin, and spirotetramat. Spinosad applied to bare-root and plug-tray transplants immediately before transplanting reduced P. gymnostoma damage in the field by >90%. Implications of using these insecticides and application strategies are discussed within the context of developing a sustainable IPM program.
The sweetpotato whitefly, Bemisia tabaci MEAM1 Gennadius, is a global pest of tomato, transmitting Tomato yellow leaf curl virus (TYLCV). Management of B. tabaci is challenging in part because of its ability to develop resistance to insecticides. Biopesticides include materials that control B. tabaci via mechanisms that do not select for resistance. Field experiments were conducted in the spring and fall of 2016 and 2017 at the University of Florida's Gulf Coast Research and Education Center in west central Florida to compare biopesticides to conventional insecticides for management of B. tabaci and TYLCV. Insecticide rotations were designed in part around the concept that conventional insecticide programs should group modes of action according to 5-wk treatment intervals, corresponding to an estimated 5-wk generation time for the pest. In 2016, when tomato was treated during the first 5-wk treatment interval with either biopesticides or neonicotinoid insecticides, insecticidal soap contributed to a reduction in whitefly egg numbers and percentageTYLCV that was comparable to results achieved with dinotefuran. In contrast, egg numbers and virus incidence in plants treated with kaolin clay tended to be numerically higher than the untreated control. In spring 2017, comparisons of biopesticides and conventional ovicides/nymphicides during the second 5-wk treatment interval showed that biopesticides can provide comparable reduction in nymph numbers to conventional insecticides. While data from these trials confirm that biopesticides can reduce numbers of whitefly eggs and nymphs, they indicate that season-long programs of the biopesticides evaluated may not reduce transmission of TYLCV below economically acceptable levels.
The billbug, Sphenophorus spp. (Coleoptera: Curculionidae), is an important pest complex in sod farms in Georgia. Larval feeding within stolons and on roots affects spring recovery of slow-growing zoysiagrass and poses a serious challenge to machine harvesting, as the damaged turfgrass rarely holds together. Little is known about major billbug species and their seasonal occurrence and abundance in Georgia sod farms, as most previous research was conducted in golf courses in the region. In 2018 and 2019, adult billbugs were sampled from five zoysiagrass sod field sites in central Georgia. Four linear pitfall traps were used per site from February to December each year, and the traps were checked at weekly intervals. The data show that >98% of the sampled billbugs were the hunting billbug, Sphenophorus venatus vestitus Chittenden, whereas the nutgrass billbug, Sphenophorus cariosus Olivier; uneven billbug, Sphenophorus inaequalis Say; and vegetable weevil, Listroderes difficilis Germain were the minor species. Seasonal billbug capture was influenced by turfgrass phenology (e.g., early-growth-stage, late-growth-stage or fully grown turfgrass). The numbers of Sphenophorus spp. collected were significantly greater in the fully grown turfgrass than in the early- or late-growth-stage turfgrasses. Significantly greater densities of billbug were found in Zoysia matrella (L.) Merrill (‘Zeon') and the Z. matrella × Zoysia pacifica (Goudswaard) M. Hotta & S. Kuroki hybrid (‘Emerald') than in the Zoysia japonica (Steudel) cultivars ‘El Toro’ and ‘Zenith'. Similar numbers of male and female billbugs were collected from the sod field sites.
Brown marmorated stink bug (Halyomorpha halys Stål) is an invasive and economically important agricultural and ornamental insect pest now established in 46 U.S. states. It was first detected in Utah in 2012 and began causing agricultural damage in 2017. Tart cherry (Prunus cerasus Linnaeus) is a major processed agricultural commodity in Utah; yet, its susceptibility to brown marmorated stink bug is unstudied. Limb cages with six brown marmorated stink bug adults, nymphs, or no brown marmorated stink bug were established in a randomized complete block design in a tart cherry orchard to determine feeding impact on different fruit developmental stages. After 1 wk of feeding, half of the fruits in each cage were removed to assess feeding intensity, and the remainder left through maturity to assess marketability and quality. Feeding by adults and nymphs between petal fall and fruit pit hardening, even at feeding pressures as low as 1.7–4.0 feeding sites per fruit, caused 100% abscission of fruits, significantly reducing marketability when compared with the control treatment. For fruits that escaped abscission and matured, few quality differences were detected among treatments, indicating that brown marmorated stink bug feeding caused minimal detectable quality loss to this processed tree fruit crop. We conclude that tart cherries are at risk of abscission with short-term brown marmorated stink bug feeding between petal fall and pit hardening when overwintered adults or F1 nymphs are present in orchards, and suggest that longer-term feeding may be necessary to cause quality and yield reductions after pit hardening.
Integrated pest management (IPM) guidelines for horticulture are typically established from years of experimental research and experience for a crop species. Ecoinformatics methods can help to quickly adapt these guidelines following major changes in growing practices. Citrus production in California is facing several major challenges, one of which is a shift away from sweet oranges [Citrus sinensis (L.) Osbeck Sapindales: Rutaceae] toward mandarins (including mostly cultivars of C. reticulata Blanco and C. clementina hort. ex Tanaka). In the absence of IPM guidelines for mandarins, growers are relying on pest information developed from oranges. We mined a database of management records from commercial growers and consultants to determine densities for four arthropod pests: cottony cushion scale (Icerya purchasi Maskell Hemiptera: Monophlebidae), citricola scale (Coccus pseudomagnoliarum Kuwana Hemiptera: Coccidae), European earwig (Forficula auricularia Linnaeus Dermaptera: Forficulidae), citrus red mite (Panonychus citri McGregor Acari: Tetranychidae), and a natural enemy, predatory mites in the genus Euseius (Congdon Acarina: Phytoseiidae). Densities of cottony cushion scale were approximately 10–40 times higher in the two most commonly grown mandarin species than in sweet oranges, suggesting this pest is reaching outbreak levels more often on mandarins. Densities of the other pests and predatory mites did not differ significantly across citrus species. This is a first step toward establishing IPM guidelines for mandarins for these pests; more research is needed to determine how arthropod densities relate to crop performance in mandarins.
The psyllid Cacopsylla chinensis (Yang & Li) (Hemiptera: Psyllidae) is a serious pest of pears in China. To determine and contrast the fitness of the psyllid on two endemic cultivars of Pyrus bretschneideri (i.e., BHXS and BSL) and two introduced cultivars of Pyrus communis (i.e., CB and CRB), we analyzed data on the development, survival, and fecundity from C. chinensis individuals reared on the four cultivars. The age-stage, two-sex life table theory was used in order to enable the inclusion of males in the analysis as well as a means of identifying the variation in developmental durations among individuals. Results indicated that C. chinensis can successfully develop and reproduce on all four pear cultivars. However, based on the lower preadult survival rate, longer preadult duration, longer total preoviposition period (TPOP), and lower fecundity that occurred on both cultivars of P. communis, these two cultivars are less favorable hosts for C. chinensis than the P. bretschneideri cultivars. The lower intrinsic rate of increase (r), finite rate of increase (λ), and net reproduction rate (R0) on CB and CRB pears showed these two introduced cultivars are more resistant to C. chinensis than the endemic BHXS and BSL pears. These resistant cultivars would be appropriate candidates for managing C. chinensis. We used the bootstrap technique to estimate the uncertainty of the population parameters (r, λ, R0, etc.), while also demonstrating that it can be used for estimating the 0.025 and 0.975 percentile confidence intervals of the age of survival rate.
Graphical Abstract
Age-stage, two-sex life table can reveal the survial and stage differentiation for assessment of differences between treatments
Integrated pest management (IPM) programs for the spotted-wing drosophila Drosophila suzukii (Diptera: Drosophilidae) rely on insecticide applications to reduce adult populations and prevent fruit infestation. Although monitoring traps are used for early D. suzukii adult detection to time the start of insecticide applications, it remains unclear whether trap counts can be used to determine the efficacy of these programs and predict the risk of fruit infestation.To address this, a 2-yr study (2016–2017) was conducted in highbush blueberries in New Jersey (USA) to interpret D. suzukii trap count variation in relation to the frequency of insecticide applications and proximity to forest habitats. We also correlated trap counts with fruit infestation and used traps to determine the maximum dispersive distance traveled by D. suzukii adults within blueberry fields by using mark-release-capture studies. Using a trapping network across nine farms, we demonstrated that insecticide applications reduce D. suzukii trap counts, but this varied according to seasonality, and that traps placed closer to forest habitats within farms had higher fly counts than those placed in farm interiors. Moreover, blueberry fields that had zero fruit infestation also had predictably lower trap counts than fields with infested fruit, and the maximum dispersive distance for D. suzukii within blueberry fields was 90 m. In summary, while D. suzukii trap counts in blueberry farms could predict the frequency of insecticide applications and fruit infestation, the predictive power of our trap data was too variable across the blueberry harvest period to make it a reliable tool.
Spotted-wing drosophila (Drosophila suzukii Matsumura) is a major pest of soft-skinned fruit and due to the low infestation tolerance for marketable fruit, growers take preventive actions to hinder spotted-wing drosophila damages. Insecticides application is one of the measures taken by growers. Although intensive spraying programs have been used to manage spotted-wing drosophila, its early infestation, rapid reproduction, and vast range of host have caused damage to still occur in fruit, including tart cherries, Prunus ceraus (Linnaeus). Therefore, there is a merit for information on insecticide's curative activity to understand whether sprays manage spotted-wing drosophila individuals within infested fruit. Tart cherry fruit were exposed to spotted-wing drosophila adults for 3 d. After this infestation period, insecticides were applied 1 and 3 d later. Small larvae, large larvae, and pupae were counted 9 d after initial infestation. A parallel set of insecticide-treated tart cherries were subjected to residue analysis. Phosmet and spinetoram were able to reduced live spotted-wing drosophila counts compared with the control at all life stages and insecticide application times, whereas zeta-cypermethrin, acetamiprid, and cyantraniliprole were less consistent in reducing spotted-wing drosophila numbers. Chromobacterium subtsugae demonstrated no curative action. Residue analysis demonstrated that zeta-cypermethrin residues mostly remained on fruit surface. Small portions of phosmet, spinetoram, and cyantraniliprole were able to penetrate fruit surfaces and move into subsurface tissues. Acetamiprid was the only compound which >47% penetrated into the fruit subsurface consistently across both years. Curative activity demonstrated in this study can provide additional tactics for spotted-wing drosophila management in tart cherry Integrated Pest Management (IPM) programs.
Rhagoletis mendax Curran (the blueberry maggot fly) is a major pest of wild blueberry. It is a direct pest of the fruit. Females lay eggs in fruit resulting in infestations of larvae unacceptable to most consumers. Three field perimeter interception tactics were tested for control of R. mendax in wild blueberry, Vaccinium angustifolium Aiton (Ericales: Ericaceae), between 2000 and 2010. We investigated field perimeter deployment of baited, insecticide-treated, green spheres (2000–2005), baited, yellow Trécé PHEROCON AM traps (2005 and 2006), and baited, Hopper Finder, sticky barrier tape (2008–2010). Only the Hopper Finder tape provided significant reduction in R. mendax adults and fruit infestation over the 3-yr field study. However, the reduction in fruit infestation compared with control plots was only 48.2 ± 7.3%, a level of reduction in damage that would be unsuitable for many commercial wild blueberry growers as a stand-alone tactic, but could be an important reduction as part of a multiple tactic IPM strategy. In addition, we constructed an agent-based computer simulation model to assess optimal trap placement between three patterns: 1) a single row of traps along field perimeter; 2) a double row of traps along the field perimeter at half the density of the single row; and 3) a grid of traps spread throughout the field but with the largest distance between traps. We found that the single row deployment pattern of traps was the best for reducing immigration of R. mendax adults into simulated fields.
In order to contrast the fitness of Thrips palmi Karny reared on two of its hosts plants, Brassica oleracea L. leaves and Phaseolus vulgaris L. beans, life tables were constructed for T. palmi individuals reared on the two hostplants at 25°C using the age–stage, two-sex life table. The durations of the egg, first instar larva, pupa, and total immature stages of T. palmi reared on P. vulgaris were significantly longer than those reared on B. oleracea, whereas the adult preoviposition period, female adult longevity, and number of oviposition days of T. palmi reared on B. oleracea were significantly longer/greater than those on P. vulgaris. The fecundity (F) of T. palmi reared on B. oleracea (159.71 offspring/female) was significantly higher than in females reared on P. vulgaris (F = 122.98 offspring/female) despite the higher proportion of female adults that emerged on P. vulgaris. Although there were no significant differences in the intrinsic rate of increase (r), finite rate (λ), net reproductive rate (R0), and mean generation time (T) between the two populations, computer projection showed that the T. palmi population will increase faster when reared on B. oleracea than on P. vulgaris. Our results demonstrate that population projection based on life table data is potentially an important tool in pest management. Furthermore, we used the bootstrap technique to demonstrate the variable effect that survival rate and fecundity at different ages can have on population parameters.
Isabel Moreira da Silva, Marcus Alvarenga Soares, Wagner de Souza Tavares, Alexandre dos Santos, José Eduardo Serrão, Antonio José Vinha Zanuncio, Carlos Frederico Wilcken, José Cola Zanuncio, Carlos Sigueyuki Sediyama
Diaphania hyalinata (Linnaeus, 1767) is one of the main pests of the cucurbit crops. Biological control and botanicals are used in integrated pest management (IPM), especially in agro-ecological agricultures where the use of synthetic pesticides is restricted. Toxicological effects of plant essential oils on target and nontarget organisms should be evaluated to determine its use in IPM. The toxicity of ginger, peppermint, oregano, and thyme essential oils to D. hyalinata eggs, larvae, and pupae and their selectivity to the parasitoid Trichospilus pupivorus and the LC50, LC99 and the relative selectivity index (RSI) of these botanicals were determined. The eggs of D. hyalinata were more sensitive to the botanicals applied than its larvae and pupae, with higher toxicity of oregano and thyme essential oils, followed by those of peppermint and ginger. Topical application was the least toxic method to D. hyalinata larvae. Ginger, peppermint, and thyme essential oils were more toxic by ingestion and oregano by contact to D. hyalinata larvae. The essential oil concentrations applied to D. hyalinata pupae prevent the emergence of adults of this insect with the oregano essential oil showing the greatest toxicity. Peppermint, ginger, thyme, and oregano essential oils were selective to T. pupivorus with RSI50 of 5.40, 1.38, 8.15, and 6.98 and RSI99 of 1.54, 2.53, 3.90, and 4.16 respectively. The ginger, peppermint, oregano, and thyme essential oils were toxic to immature D. hyalinata and selective to T. pupivorus females presenting potential as an alternative control in the IPM of this pest in Cucurbitaceae crops.
Rhynchophorus palmarum (L.) is an invasive pest responsible for killing thousands of ornamental Canary Islands date palms (Phoenix canariensis Chabaud) in San Diego County, CA. Two field experiments were conducted to compare the attractiveness of six different baits and two trap types. The tested baits were dates + water; dates + water + Saccharomyces cerevisiae; dates + water + S. bayanus; dates + water + S. pastorianus; 15% sugarcane molasses water solution mixed with 3% paraffinic oil, and a no bait control treatment. The two traps tested were white bucket traps (hanging 1.5 m above the ground and set on the ground) and black cone shaped Picusan traps (set on ground only). All traps were loaded with commercially available R. palmarum aggregation pheromone and the synergist ethyl acetate. Differences in weevil capture rates were observed across bait and trap types. Weevil captures were almost five times greater in Picusan traps compared to bucket traps that were hanging or placed on the ground. Adding dates and water alone or combined with S. cerevisiae to traps increased weevil captures compared to other baits and controls. Trap and bait types affected bycatch of nontarget arthropod species. In general, spiders, earwigs, and carabid beetles were most commonly recovered from Picusan traps, regardless of bait type. Scarab beetles, moths, and flies were found more frequently in bucket traps baited with molasses or dates mixed with S. bayanus. No effects of bait and trap type were associated with bycatch species richness.
Adult ambrosia beetles (Coleoptera: Curculionidae: Scolytinae) bore into ornamental nursery trees resulting in trunk vascular tissue damage, which can potentially kill trees. Ambrosia beetle exposure to surface-applied insecticides is minimal after internal trunk galleries are formed, so effective management requires insecticide treatments to be applied near the time of infestation or to have residual activity on the bark. Tree trunk sections (bolts) were used to determine the effect of field aging or irrigation (i.e., simulated rainfall weathering) on permethrin residual activity against ambrosia beetles. In all experiments, 30-cm-long bolts from Liriodendron tulipifera L. (Magnoliales: Magnoliaceae) were hollowed and filled with 70% ethanol at field deployment to induce ambrosia beetle attacks over a 2-wk period. To evaluate insecticide residual activity, permethrin was sprayed onto tree bolts at 0, 8, 17, or 24 d before ethanol addition, and then bolts were deployed along a wooded border in fall 2017 and spring 2018. Tree bolts with permethrin residues ≤17 d old had significantly fewer ambrosia beetle attacks than bolts with 24-d-old residues or the non-permethrin-treated control bolts. To evaluate simulated rainfall weathering, permethrin was applied to tree bolts 8 or 22 d before ethanol (spring 2018) or 10 or 24 d before ethanol (fall 2018) with half of the bolts receiving regular irrigation events. Irrigation had no significant effect on permethrin residual activity against ambrosia beetles during either test. This study determined ambrosia beetle control was affected by permethrin residue age more than simulated rainfall weathering, and a reapplication interval of ≤17 d maximized beetle control.
We evaluated the effect of postharvest cold storage temperature (0.0–2.2°C) and duration (3–5 d) on pupal emergence of single life stage populations of laboratory-generated Drosophila suzukii (Matsumura) and mixed-age populations collected in the field from blackberries, blueberries, strawberries, and raspberries. For field-infested fruit, cold storage at any temperature and duration resulted in less pupal emergence compared with the control held at 20°C, but D. suzukii subjected to higher temperatures and shorter storage durations in caneberries had higher survival. When a single life stage of D. suzukii was exposed to cold storage, pupal emergence was significantly reduced across all fruit types held at 0°C and for most life stages and fruit types held at 1.5 and 2.2°C, dependent on the substrate. Freshly laid eggs exposed to cold storage produced the lowest pupal emergence. Our results suggest using cold storage treatment is an effective postharvest management strategy for small-fruit growers to use on-farm as part of an integrated program to manage D. suzukii infestation. An economic assessment was made to examine the profit implications of an investment in cold storage units to counter pest pressure. Results suggest that investment in a cold storage unit would breakeven in about 4 yr. On farms that already have cold storage installed, we estimated a $0.11/kg decrease in blueberry market price for holding fruit for 3 d. Together, this cost assessment will provide growers with the knowledge to make decisions based on infestation risk and the seasonal sale price of blueberries.
The use of essential oil components (EOCs) against the German cockroach, Blattella germanica (L.), has increasingly received attention from researchers. However, the determination of lethal doses/concentrations alone does not provide enough information on the range of biological effects of these EOCs. To improve our understanding of the potential effects of EOCs, we examined biological parameters of B. germanica exposed to sublethal EOCs formulated in gels. This study employed superabsorbent polymer (SAP) gel to prolong bioavailability of limonene, carvacrol, and β-thujaplicin, and evaluated how these EOCs shape biological parameters of B. germanica. Overall, median survival days ranged from 57 to 69.5 d for males and 73 to 99 d for females. The survival day ranking for the EOCs was limonene > β-thujaplicin > carvacrol. Carvacrol and β-thujaplicin gels reduced male longevity by at least 34 and 39%, respectively, while limonene had no effect. The longevity of females was reduced by limonene, but not by carvacrol and β-thujaplicin gels. EOCs significantly suppressed overall adult females' reproductive period, oothecal hatchability, reduced fecundity, and interoothecal period, but not the number of oothecae formed and egg incubation period. The preoviposition period (mean: 2–25 d) ranking was limonene < carvacrol < β-thujaplicin. Based on these results, limonene, carvacrol, and β-thujaplicin in SAP gels show promising potential to reduce adult male survival/longevity, suppress egg hatchability and female fecundity, and delay the interoothecal period. These findings may represent the basis for the practical use of EOCs as a tactic in integrated pest management systems for B. germanica.
Colonies of western drywood termites, Incisitermes minor (Hagen) (Blattodea: Kalotermitidae), are difficult to detect and treat due to their cryptic nature. The use of heated air to create lethal temperatures within infested wood serves as a nonchemical treatment option targeting whole structure or large portions of the structure. However, the presence of hard-to-heat areas and potential risk of damage for heat-sensitive items are recognized as important challenges. Here, we tested if a localized injection of volatile essential oil could be utilized to address the heat sink issue, potentially increasing the overall efficiency of heat treatments against drywood termites. Artificially infested wooden blocks were placed in several locations of the test building, and heat treatments were conducted. For the treatment group, a small amount of essential oil (methyl salicylate) was added in the blocks prior to the heat treatment. All blocks placed in uninsulated wall voids had 92–100% termite mortality by day 7. However, the presence of a large concrete wall in the subarea hindered heating of blocks therein, resulting 36–44% mortality by day 7 when there was no essential oil treatment. Incorporation of the essential oil substantially increased the control efficacy for the subarea, resulting in more than 90% mortality.This approach might also be helpful in reducing the risk of potential heat damage during heat treatment without compromising its control efficacy.
We conducted maximum dose bioassays of insecticide for the control of diamondback moth (DBM), Plutella xylostella (Linnaeus), in cole crops, from 2016 to 2019 at several commercial locations in Georgia and Florida. The nominal maximum dose was defined as the highest labeled rate of an insecticide at the beginning of the survey in the equivalent of 935 liters/ha dilution. The results indicated low insecticide efficacy for high labeled rates of the following insecticides by common name (Insecticide Resistance Action Committee group number in parentheses). Our 4-yr survey identified very low levels of DBM larval control (<47%) by lambda-cyhalothrin (3), methoxyfenozide (18), pyriproxyfen (7C), novaluron (15), bifenthrin (3), chlorantraniliprole (28), indoxacarb (22A), and methomyl (1A). The best products for DBM control (>74%) listed in decreasing average levels of efficacy were naled (1B), cyclaniliprole (28), tolfenpyrad (21A), emamectin benzoate (6), and cyantraniliprole (28). Intermediate levels of control (61–71%) were obtained with Bacillus thuringiensis subspecies aizawai (11A), Bacillus thuringiensis, subsp. kurstaki, strain ABTS-351 (11A), and spinetoram (5). This rapid bioassay provided the grower with a ranking of insecticide efficacy for the control the DBM population for that farm site. These data allowed growers to make an informed decision on control quickly and plan for resistance management rotations for DBM that season.
Western corn rootworm, Diabrotica virgifera virgifera LeConte, is a serious pest of corn and is often managed with transgenic corn producing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt).This pest has developed field-evolved resistance to all commercially available Bt traits, beginning with Cry3Bb1 in 2009. Fitness costs may accompany Bt resistance, where individuals with alleles for Bt resistance have reduced fitness on non-Bt corn compared to Bt-susceptible individuals. In conjunction with non-Bt refuges, fitness costs can delay the evolution of Bt resistance. Importantly, ecological factors may affect the presence and magnitude of fitness costs. For western corn rootworm, available data suggest that fitness costs of Bt resistance may be present in some cases. Using two Cry3Bb1-resistant western corn rootworm strains (Hopkinton and Cresco), a fitness-cost experiment was performed by rearing rootworm in the absence of Bt for six generations to test for fitness costs of Cry3Bb1 resistance and the effect of larval rearing density on fitness costs. Fitness costs were detected for both strains; however, strains were still resistant to Cry3Bb1 corn at the end of the experiment. Cresco experienced a greater loss of resistance at low versus high density, but no effect of density was detected in Hopkinton. Our study shows that fitness costs can accompany Bt resistance in western corn rootworm and may be more pronounced under low larval density. Even though fitness costs were present, it appears that rootworm populations may remain resistant to Cry3Bb1 corn for years after resistance has evolved.
The emerald ash borer (EAB), Agrilus planipennis (Fairmaire), is the most destructive invasive insect species of ash (Fraxinus spp.) in North America. An accurate method for early detection of this noxious insect pest is indispensable to providing adequate warning of A. planipennis infestation. A loop-mediated isothermal amplification (LAMP) assay (EAB-LAMP) was developed based on mitochondrial cytochrome c oxidase subunit I (COI) gene. The EAB-LAMP required only 30 min at 65°C to amplify A. planipennis DNA from specimens collected from geographically distinct locations.There was no cross-reactivity with other Agrilus and insect species.The developed EAB-LAMP differentially detected traces of A. planipennis genome (COI) within frass from various Fraxinus species. EAB-LAMP was also able to distinguish among A. planipennis DNA and other Agrilus species and nontarget insect species in trap captures. By detecting A. planipennis DNA in two additional trap captures (in situ), the EAB-LAMP was more sensitive and reliable than visual inspection. We tested the quantitative nature of the assay by evaluating pooled trap samples and demonstrated that the EAB-LAMP was capable of functioning optimally using a pool size of at least five individual trap samples. This potentially circumvents the need to perform large-scale individual analysis for processing trap samples. Considering its performance, specificity, sensitivity, and repeatability, the developed EAB-LAMP could be a valuable tool to support strategy and operation of large-scale surveillance for A. planipennis and could profitably be used in routine monitoring programs for effective management of A. planipennis.
Tephritid fruit flies are highly successful invaders and some—such as the Mediterranean fruit fly, Ceratitis capitata (Wiedemann)—are able to adapt to a large range of crops. Biosecurity controls require that shipments of produce are ensured to be pest-free, which is increasingly difficult due to the ban of key pesticides. Instead, stress-based strategies including controlled atmosphere, temperature, and irradiation can be used to eradicate flies inside products. However, unlike pesticide science, we do not yet have a robust scientific approach to measure cost-effectively whether a sufficiently lethal stress has been delivered and understand what this stress does to the biology of the pest. The latter is crucial as it would enable a combination of stresses targeting multiple molecular pathways and thus allow for lower doses of each to achieve higher lethality and reduce the development of resistance. Using heat as an example, this is the first study investigating the molecular stress response to heat in Tephritidae. Using a novel setup delivering measured doses of heat on C. capitata larvae and a high-density 11 timepoint gene expression experiment, we identified key components of lethal heat-stress response. While unraveling the complete molecular mechanism of fruit fly response to lethal stress would be a long-term project, this work curates and develops 31 potential biomarkers to assess whether sufficient lethal stress has been delivered. Further, as these protocols are straightforward and less expensive than other—omic approaches, our studies and approach will assist other researchers working on stress response.
Many genetic studies in insects require sex identification of individuals in all developmental stages. The most common sex chromosome system in lepidopterans is WZ/ZZ; the W chromosome is present only in females. Based on two W chromosome-specific short sequences (CpW2 and CpW5) described in Cydia pomonella (L.) (Lepidoptera: Tortricidae), we identified homologous female-specific sequences in Lobesia botrana Den. & Schiff, a polyphagous and very harmful species present in Chile since 2008. From this starting point, we extended the sequence information using the inverse PCR method, identifying the first W-specific sequences described up to now for the moth. Finally, we developed a duplex PCR method for rapid and sensitive determination of sex in L. botrana from larva to adult.The method showed a detection limit of 1 pg of genomic DNA; a blind panel of samples exhibited exact correspondence with the morphological identification. These results will be very useful for studies requiring sex-specific analyses at any developmental stage, contributing also to the understanding of gene expression in the insect, as well as to the eventual development of control protocols against the moth, such as the development of genetic sexing strains for the implementation of the sterile insect technique.
The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera; Aleyrodidae), and greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae), are highly problematic plant pests and virus vectors with worldwide distributions. Identification of whitefly species is typically accomplished by observation of distinct morphological characters; however, because of morphological inconsistency and indistinguishability, the discrimination of B. tabaci species variants is dependent on molecular techniques based on genetic differences. New assays were designed for the detection of B. tabaci A, B, and Q mitotype groups, and T. vaporariorum. Specific primer sets were designed for amplification of the mitochondrial cytochrome c oxidase I gene of the four targets to perform in end-point PCR, real-time PCR coupled to high-resolution melting analysis (HRM), and the isothermal helicase-dependent amplification (HDA). Primer specificities were validated using end-point PCR, then tested in HRM and HDA. Bemisia tabaci A, B, and Q mitotypes, and T. vaporariorumtargeted primer sets discriminately amplified specimens of different populations within their target whitefly group. These tests provide three novel discrimination assays for the high-consequence, exotic B. tabaci B and Q groups, along with the native B. tabaci A group and T. vaporariorum.
Spodoptera frugiperda (J.E. Smith) can attack cowpea plants (Vigna unguiculata [L.] Walp.) (Fabales: Fabaceae) in any growth stage but attacks primarily in the first days after plant emergence, when the plants are more sensitive to defoliation. This study was carried out to evaluate the resistance to S. frugiperda in six cowpea genotypes, of which four were cultivars (BRS Tapahium, BRS Tumucumaque, Fradinho, and Miranda), and two were landraces (Juti and Nioaque), in three different experiments. In the first experiment, adult emergence, adult weight, adult longevity, larva to adult period, numbers of injured leaves per plant canopy (upper, middle, and lower), and reduction in plant dry matter were assessed. In the second experiment, the oviposition preference of S. frugiperda for the different cowpea genotypes was evaluated under free-choice conditions. In the third experiment, levels of peroxidase (POD), superoxide dismutase (SOD), and protein content in cowpea leaves were assessed. Insects fed on landrace Juti plants showed low adult emergence, yielded a lower fitness index, and caused less plant dry matter reduction. In addition, plants of landrace Juti were less preferred for oviposition. Overall, Juti plants showed higher resistance levels in all three resistance categories and higher levels of POD and SOD in S. frugiperda injured leaves, in addition to a lower protein content. Juti will be tested in field conditions, followed by molecular characterization. This will provide additional information about its potential as an S. frugiperda resistance source in plant breeding programs.
Over the recent years, Atractomorpha sinensis I. Bolivar, has emerged as an important agricultural pest in China. However, the biological characteristics of A. sinensis remain largely unknown, which can hinder the prediction of its population dynamics. Thus, understanding the impact of temperature on the developmental period of A. sinensis is crucial to predict its population dynamics. The biological characteristics of A. sinensis were systematically observed at five different temperatures (16, 20, 24, 28, and 32°C) using the age-stage, two-sex life table method. The results demonstrated that the developmental period, preadult time, adult longevity, adult preoviposition period, and total preoviposition period were significantly reduced when the temperature was elevated from 16 to 32°C. The developmental threshold temperatures of egg, nymph, preoviposition period, female adult, male adult, and generation were 9.14, 10.44, 12.53, 10.97, 12.47, and 10.58°C, respectively, with the corresponding effective accumulated temperatures of 452.31, 575.99, 169.58, 528.13, 340.81, and 1447.95 degree-days. With an increase in temperature, the intrinsic rate of increase (r) and finite rate of increase (λ) were increased, while the mean generation time (T) was shortened. The optimal values of net reproductive rate (R0= 73.00 offspring) and fecundity (244.55 eggs) were determined at 24°C. Similarly, the population trend index (I) of A. sinensis was found to be highest at 24°C. Our findings indicate that A. sinensis has the greatest rate of population growth at 24°C, which can provide a scientific basis for predicting the in-field population dynamics of A. sinensis.
Three strains of fungi belonging to the genus Metarhizium Sorokīn (ARSEF 4556, ARSEF 3297, native strain) were assayed against adults and nymphs of the Neotropical brown stinkbug Euschistus heros (F.) and the green-belly stinkbug Dichelops furcatus (F.).The most virulent strain, ARSEF 4556, caused over 90% mortality. The average survival time of the second and fifth instar nymphs and adults following immersion in 1 × 108 conidia ml–1 was 4.8, 5.7, and 5.2 d, respectively. The second instar nymphs were more susceptible than the adults. The LC50 values and median survival times for second instar and adult E. heros were 1.6 × 107 and 3.1 × 107 conidia ml–1 and 6 and 8 d, respectively. Eggs of E. heros and the closely related stinkbug, D. furcatus, were highly susceptible to ARSEF 4556 with the mean mortality of eggs immersed in 1 × 108 conidia ml–1 being 77.4% and 89.7%, respectively. The strain 3297 showed also good aptitudes for stinkbugs control with mortalities higher than 80% against nymphs and adults and eggs mortalities of 75.5% for E. heros and 79.6% for D. furcatus. This study has shown that it is possible to have a two-pronged control strategy, targeting adults and to reduce oviposition and targeting egg clusters to prevent emergence and dispersal of nymphs. Besides early instars of nymphs have been shown to be more susceptible to the fungal strains than late instars and adults.
An ovigeny index, which is the initial egg load divided by the potential lifetime fecundity, was developed for the parasitoid, Gronotoma micromorpha (Perkins), on the host Liriomyza trifolii (Burgess) (Diptera: Agromyzidae). The value obtained for the index was estimated to be 0.54, based on the initial egg load (mean ± SD: 41.0 ± 13.5) observed in the present study and the lifetime fecundity (75.6 ± 32.6) estimated in a previous study. Gronotoma micromorpha was previously regarded as being strictly pro-ovigenic based on daily progeny production data; however, the findings of the present study showed that this parasitoid is prosynovigenic. A 3-d cycle in daily progeny production was previously reported to occur during the lifetime of adult females (6.6 d) in this species. The present study showed that the initial egg load is nearly equal to the total number of eggs deposited in 3 d following adult emergence.Therefore, it is considered that the mature eggs stored during the pupal stage are deposited during the first 3 d after adult emergence and that egg maturation in the adult stage starts on day 1 and requires 3 d to complete in G. micromorpha. To clarify the relationship between the daily progeny production and the initial egg load, a reproductive cycle consisting of maturation, deposition, and depletion of eggs is proposed. In G. micromorpha, the reproductive cycle is considered to occur twice during the female lifetime. Furthermore, it is proposed that other prosynovigenic parasitoid species have a similar reproductive cycle.
In recent years, black soldier fly, Hermetia illucens (L.), larvae have attracted increasing attention because of their high capacity for bioconversion of diverse organic material into high-quality protein and lipids. Although previous studies have focused on optimization of breeding conditions, such as the acceptance of substrates, and temperatures and moisture contents, little is known about light-dependent adult development. Artificial light sources are important to commercial H. illucens breeding, especially at latitudes with short days in autumn and winter months. We examined how 3,000, 4,000, and 6,500 K color temperatures affect aspects of oviposition. Mating occurred under all of the broad spectrum light-emitting diode panels, resulting in fertilized egg clusters. Oviposition lasted up to 15 d, while the shortest oviposition period, in the 3,000 K light treatment, was 2 d. Total oviposition performance and oviposition period were not affected by the light treatments. Oviposition peaked 1–7 d after eggs were first deposited. The time until oviposition peaked was positively correlated with increasing color temperature.
Low tunnels covered with spun-bonded fabric (row covers) provide season extension for vegetable production and also afford a physical barrier against airborne insects and other non-soil pests. Brussels sprouts, Brassica oleracea L. group Gemmifera (Brassicaceae), is a popular vegetable in local markets in Virginia; however, unprotected field production is severely affected by insect pest infestation.This study's objective was to determine the level of protection low tunnels provide against insect infestation and leaf herbivory injury.The experiment was conducted at the Virginia Tech Eastern Shore Agricultural Research and Extension Center in Painter,Virginia. The experimental design was split-plot with polyethylene soil mulches (white or black) as whole plot factors and production systems (low tunnel or open field) as subplot factors. In this study, low tunnels reduced insect infestation and chewing herbivory leaf injury to Brussels sprouts. Compared to an unprotected open field, infestations of lepidopteran insects and harlequin bug, Murgantia histrionica (Hahn) (Hemiptera: Pentatomidae) were reduced on plants under low tunnels. However, aphids (Hemiptera: Aphidae) infestation occurred under low tunnels in fall.There was no effect of color mulches (white or black) and no interaction between tunnel and mulch color on insect infestation and chewing injury. Fewer insect infestations and feeding injury indicate that low tunnels can be an effective management tool for sustainable vegetable production.
The three-cornered alfalfa hopper, Spissistilus festinus (Say), was shown to transmit Grapevine red blotch virus (GRBV), the causative agent for Grapevine red blotch disease, in a greenhouse study on grapes. GRBV is a major concern of wine grape growers due to its economic impact on wine quality. Plants in the family Fabaceae are preferred hosts of S. festinus and are commonly planted as cover crops or present in a vineyard's native vegetation. In late winter, during grapevine dormancy, S. festinus migrate into vineyards to feed and reproduce on these cover crop and weed hosts. Tilling vineyard floor vegetation provides growers an opportunity to disrupt the life cycle of early instars that are relatively immobile, reducing the S. festinus first-generation population. Nymphal presence is difficult to detect. First through third instars were not detected in sweep net samples in a 2-yr weekly sampling study, whereas fourth and fifth instars were first found on the same sample date as emerging adults. A degree-day model was developed and successfully predicted when early S. festinus instars are present in the vineyard to aid in exploiting the time period when S. festinus is most susceptible to cultural control measures.
Asian citrus psyllid, Diaphorina citri Kuwayama, negatively impacts the citrus industry as it transmits Candidatus Liberibacter spp., the causal agent of citrus greening. Monitoring D. citri population levels is critical for management of vectors and citrus greening and is usually through use of yellow sticky traps. In our study, use of odors, odor blends, and visual targets were evaluated to determine whether attraction to yellow sticky traps could be improved. Methyl salicylate consistently increased D. citri attraction to decoy yellow but not to other yellow or yellow/green traps. Addition of a visual target did not enhance attraction to sticky traps. While several chemical blends were evaluated, they did not increase psyllid attraction to decoy yellow traps.The increased attraction to traps with methyl salicylate is promising and may contribute to trapping efficiency under field conditions.
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