The development of Spodoptera frugiperda (Lepidoptera: Noctuidae) on 8 major crops in Japan and its oviposition preference for maize and soybean.

The fall armyworm, Spodoptera frugiperda (J.E. Smith), is a polyphagous pest native to the Americas. It first invaded Japan in July 2019, and the most damaged crop in this region to date has been maize. To determine pest control strategies, the range of crops potentially damaged by S. frugiperda in Japan should be identified. In this study, the effects of 8 major crops in Japan-forage maize Zea mays subsp. mays, sugarcane Saccharum officinarum, forage and edible rice Oryza sativa subsp. japonica, soybean Glycine max, eggplant Solanum melongena, green bell pepper Capsicum annuum var. grossum, sweet potato Ipomoea batatas, and taro Colocasia esculenta-on the development of S. frugiperda were analyzed by feeding leaves of domestic cultivars during larval stage. Spodoptera frugiperda developed from hatching to adulthood and laid eggs in all 7 treatments, except for taro. However, among them, only soybean showed comparable developmental suitability to maize. Therefore, its oviposition preference for maize and soybean was examined using further nonchoice and choice tests. In the nonchoice test, the number of egg masses oviposited for 3 days on soybean plants was significantly less than that on test containers, while that on maize was comparable to that on containers. These findings can explain partly why major damage has been limited to maize in Japan.

RNAi of yellow-y, required for normal cuticle pigmentation, impairs courtship behavior and oviposition in the German cockroach (Blattella germanica).

The insect cuticle plays a key role in maintaining the insect's physiological function and behavior. Herein, the yellow-y protein is required to produce black melanin, and is expressed in a pattern that correlates with the distribution of this pigment. However, yellow-y can also have other functions, for instance, in insect behavior, but not much is known. In this study, we have studied the yellow-y gene in one important model and pest species, namely the German cockroach (Blattella germanica), which is to our knowledge the first time reported. In essence, we identified the yellow-y gene (BgY-y) and characterized its function by using RNA interference (RNAi). Silencing of BgY-y gene led to different developmental abnormalities (body weight and wings) in both genders. Specifically, there was an abundant decrease in melanin, turning the body color in pale yellow and the cuticle softer and more transparent. Interestingly, we also observed that the knockdown of BgY-y impaired the male cockroaches to display a weaker response to female-emitted contact sex pheromones, and also that the oviposition ability was weakened in the RNAi females. This study comprehensively analyzed the biological functions of the yellow-y gene in German cockroaches from the perspectives of development, body color, courtship behavior and oviposition, and as a consequence, this may opens new avenues to explore it as a novel pest control gene.

Effect of salinity on the oviposition and growth of Ochlerotatus togoi.

Ochlerotatus togoi is a salt-tolerant euryhaline mosquito that lays its eggs in rock pools. Although it is a pest that can transmit flaviviruses and filarial worms to humans, ecological studies have not been previously conducted because of its limited habitat. However, rising sea levels have created a more favorable environment for Oc. togoi, increasing the risk of Oc. togoi-borne diseases. We examined the oviposition and growth rates of Oc. togoi at 0-35 psu to obtain ecological data. It exhibited the highest oviposition preference at 0 psu; however, the hatching rate was highest at 10 psu, the pupation rate was highest at 25 psu, and the emergence rate was highest at 5 psu. Oc. togoi showed the highest rate of growth into adults at 25 psu. The results were assessed using Mann-Whitney U and Kruskal-Wallis H tests (post hoc test: Bonferroni), and a regression equation was generated for the incidence of adult Oc. togoi based on the change in salinity (y = -14.318 + 9.821x; y = adult incidence rate; x = salinity). The oviposition habits and developmental conditions of Oc. togoi were confirmed, and the incidence of Oc. togoi based on changes in sea level and ocean salinity was predicted. The results of this study will be useful for controlling salt-tolerant vectors and responding to vector-borne diseases.

Oviposition-Deterrent Effect of a High-Quality Natural Zeolite on the Olive Fruit Fly Bactrocera oleae, under Different Conditions of Temperature and Relative Humidity.

In recent years, the number of available chemical pesticides has been dramatically reduced, urging the need for the discovery of alternatives to chemical pesticide products such as, among others, natural zeolites (zeolitic rocks). We determined the mineralogical and chemical composition of a specific and continuous layer of zeolitic rock sample (ZeotP) from Petrota, Evros, Greece, and evaluated its oviposition-deterrent effect on the olive fruit fly Bactrocera oleae Gmelin (Diptera: Terphritidae). The tested natural zeolite contained 70 wt. % clinoptilolite, 18 wt. % amorphous material, 7 wt. % feldspars, 4 wt. % cristobalite, and 1 wt. % quartz. We tested the oviposition-deterrent effect of ZeotP mixed or not with an emulsifier adjuvant, NU-FILM-P®, in water and applied it to the surface of olive fruits. The ZeotP oviposition-deterrent effect on the olive fly was very high under a series of tested temperatures (17 °C, 20 °C, 25 °C, and 30 °C) and RHs (23%, 33%, 55%, 75%, and 94%). In addition, the ZeotP residual deterrent effect after equable water spraying was high, like the respective effect of the pyrethroid insecticide Decis® (deltamethrin). Our results may contribute to the effective control of the olive fruit fly using an alternative to chemical pesticides: natural zeolite (zeolitic rocks) products.

Accessory gland protein regulates pairing process and oviposition in the subterranean termite Reticulitermes chinensis after swarming.

Swarming and pairing behaviors are significant to population dispersal of termites. Tandem running is a key process in pairing behavior of dealates to find a mate. Succinylation can lead to significant changes in protein structure and function, which is widely involved in metabolism and behavior regulation in many organisms. However, whether succinylation modification regulates termites' tandem running is currently unknown. In this research, we performed quantitative modified proteomics of the subterranean termite Reticulitermes chinensis Snyder before and after alate swarming. The succinylation levels of accessory gland protein (ACP) were significantly altered after alate swarming. We found that ACP is enriched in male accessory gland and female oocytes of termites. The acetylation and succinylation sites of ACP affected tandem running of dealates. The transcriptome and metabolome analyses of alates injected with ACP and its mutant proteins showed that ß-alanine metabolism pathway was the major downstream pathway of ACP. Silencing the significantly differentially expressed genes in the ß-alanine metabolic pathway (acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyisobutyrate dehydrogenase, methylmalonate-semialdehyde dehydrogenase) suppressed tandem running and altered oviposition of paired dealates. These findings demonstrate that protein translation modification is an important regulator of tandem running behavior of termites, which implies that the succinylation and acetylation modification sites of ACP could be potential targets for insecticide action. Our research offers a potential approach for developing novel dispersal inhibitors against social insect pests.

miR-2940-1 is involved in the circadian regulation of oviposition in Aedes albopictus.

The vast majority of all global species have circadian rhythm cycles that allow them to adapt to natural environments. These regular rhythms are regulated by core clock genes and recent studies have also implicated roles for microRNAs in this regulation. Oviposition is an important circadian behavior in the reproductive cycle of insect vectors of diseases, and little is known about the rhythm or its regulation in mosquitoes. Aedes albopictus is a diurnal mosquito that transmits arboviruses and is the major cause of outbreaks of dengue fever in China. We analyzed the oviposition rhythm patterns of A. albopictus under different light/dark conditions and show that the mosquitoes have an oviposition peak between zeitgeber time 9 (ZT 9) and ZT 12. Furthermore, the antagomir-mediated knockdown of expression of the microRNA miR-2940-1 affected the oviposition rhythm of A. albopictus. These data support the conclusion that miR-2940-1 is involved in the regulation of oviposition rhythm in A. albopictus and provide a foundation for using oviposition rhythms as a new target for vector mosquito control.

First evidence of late-acting self-incompatibility in the Aristolochiaceae.

Most Aristolochiaceae species studied so far are from temperate regions, bearing self-compatible protogynous trap flowers. Although self-incompatibility has been suggested for tropical species, the causes of self-sterility in this family remain unknown. To fill this gap, we studied the pollination of the tropical Aristolochia esperanzae, including the physical and physiological anti-selfing mechanisms. Floral visitors trapped inside flowers were collected to determine the pollinators. Protogyny was characterized by observing the temporal expression of sexual phases and stigmatic receptivity tests. The breeding system was investigated using hand-pollination treatments. Pollen tube growth was observed using epifluorescence to identify the self-incompatibility mechanism. Flies were the most frequent visitors found inside A. esperanzae trap flowers, with individuals from the family Ulidiidae being potential pollinators since they carried pollen. The characteristic flower odour and presence of larvae indicate that A. esperanzae deceives flies through oviposition-site mimicry. Although this species showed incomplete protogyny, stigmatic receptivity decreased during the male phase, avoiding self-pollination. Fruits developed only after cross- and open pollination, indicating that the population is non-autonomous, non-apomictic, and self-sterile. This occurred through a delay in the growth of geitonogamous pollen tubes to the ovary and lower ovule penetration, indicating a late-acting self-incompatibility mechanism. Our findings expand the number of families in which late-acting self-incompatibility has been reported, demonstrating that it is more widespread than previously thought, especially when considering less-studied tropical species among the basal angiosperms.

How conspecific and allospecific eggs and larvae drive oviposition preference in Drosophila.

Where to lay the eggs is a crucial decision for females as it influences the success of their offspring. Female flies prefer to lay eggs on food already occupied and consumed by larvae, which facilitates social feeding, but potentially could also lead to detrimental interactions between species. Whether females can modulate their attraction to cues associated with different species is unknown. Here, we analyzed the chemical profiles of eggs and larvae of 16 Drosophila species, and tested whether Drosophila flies would be attracted to larvae-treated food or food with eggs from 6 different Drosophila species. The chemical analyses revealed that larval profiles from different species are strongly overlapping, while egg profiles exhibit significant species specificity. Correspondingly, female flies preferred to lay eggs where they detected whatever species' larval cues, while we found a significant oviposition preference only for eggs of some species but not others. Our findings suggest that both larval and egg cues present at a given substrate can drive oviposition preference in female flies.

Leaf trichome-mediated predator effects on the distribution of herbivorous mites within a kidney bean plant.

Some predators prefer to settle on leaf patches with microstructures (e.g., trichomes and domatia), leaving traces on the patches. Herbivorous arthropods, in turn, select leaf patches in response to these traces left by predators. It remains unclear whether traces of predators on leaf patches affect the distribution of herbivorous prey within plants through plant microstructure. Therefore, we examined the distribution of herbivorous mite (Tetranychus urticae) and predatory mite (Phytoseiulus persimilis) by investigating their oviposition pattern. We used a kidney bean plant (Phaseolus vulgaris) with two expanded primary leaves and the first trifoliate leaf, focusing on leaf trichomes as the microstructure. The density of trichomes was higher on the first trifoliate leaf than on the primary leaves and on the abaxial surface of the leaves than on the adaxial surface. Adult female P. persimilis laid more eggs on the first trifoliate leaf to the primary leaves. Although adult female T. urticae preferred to oviposit on the abaxial surface of primary leaves, previous exposure of plants to predators diminished this preference. The altered egg distribution would be a response to the traces of P. persimilis rather than eggs of P. persimilis. Our findings indicate that T. urticae reproduces on leaf patches with traces of predators without altering their oviposition preference. Given that the presence of predator traces is known to reduce the reproduction of T. urticae, it may have a substantial effect on the population of T. urticae in the next generations on kidney bean plants.

Arboreal or terrestrial: Oviposition site of Zhangixalus frogs affects the thermal function of foam nests.

Temperature is essential for the survival and development of eggs. Some anurans have evolved and developed foam nesting traits, with thermal insulation considered to be among their functions. Foam-nesting frogs tend to exhibit reproductive plasticity. For example, they oviposit on both trees and the ground. How such plasticity affects foam nest function is of major relevance and is likely related to the adaptation of foam nesting frogs. However, this has not been well studied. In this study, we examined the interaction between foam nest site, foam nest function, and egg fate using the Japanese green tree frog, Zhangixalus arboreus, and analysed how nest site differences (arboreal or terrestrial) affect the thermal function of foam nests. We compared the thermal functions of foam nests between arboreal and terrestrial oviposition sites of Z. arboreus. We artificially replaced half of the arboreal nests with terrestrial environments and recorded temperature in and outside of the experimental terrestrial nest and original arboreal nests. We also examined egg survival and hatching rates for all the nests. The results indicated superior heat insulation in terrestrial nests, with warmer temperatures inside than outside the nests, especially at night, which led to a high egg survival rate. Therefore, terrestrial ovipositing should be valid under cold weather conditions. This may be related to the evolutionary history of oviposition site plasticity of this genus, which originally had an arboreal oviposition trait but evolved into terrestrial site use owing to global cooling. Our novel insights into the evolution and adaptivity of foam nesting and oviposition site use in Z. arboreus make an important contribution to animal ecology.

METARHIZIUM ANISOPLIAE SENSU LATO NATIVE TO LIVESTOCK SOILS CAUSES HIGH MORTALITY ON RHIPICEPHALUS MICROPLUS LARVAE, ADULTS AND AFFECTS THEIR REPRODUCTION.

The acaricidal effect of 14 strains of Metarhizium anisopliae sensu lato isolated from soil of livestock farms in the Mexican tropics was evaluated against larvae and engorged females, and during the laying and hatching of eggs of Rhipicephalus microplus (Ixodida: Ixodidae). For each fungal strain, the larvae mortality percentage was evaluated through a larval immersion test, while the reproductive efficiency indices in engorged females were measured using adult immersion tests at a dose of 1 × 108 conidia/ml. All strains of M. anisopliae (s.l.) proved to be highly effective against R. microplus larvae (66-100%) and engorged females (100%). The strains also showed a good effect in inhibiting egg laying (16.45-56.38%) and a moderate effect in decreasing egg hatching (5.24-32.68%). Two strains demonstrated to be effective against all development phases of R. microplus in an integrated manner.

A sweet tooth makes a fly a pest.

The major insect pest of soft and stone fruits, the spotted-wing drosophila, Drosophila suzukii, has evolved a greater preference for laying eggs on ripe fruits over fermented ones. In a recent study, Cavey et al. found that higher responsiveness to low sugar concentrations has had an important role in this evolutionary shift in egg-laying behavior.

Sex peptide receptor is not required for refractoriness to remating or induction of egg laying in Aedes aegypti.

Across diverse insect taxa, the behavior and physiology of females dramatically changes after mating-processes largely triggered by the transfer of seminal proteins from their mates. In the vinegar fly Drosophila melanogaster, the seminal protein sex peptide (SP) decreases the likelihood of female flies remating and causes additional behavioral and physiological changes that promote fertility including increasing egg production. Although SP is only found in the Drosophila genus, its receptor, sex peptide receptor (SPR), is the widely conserved myoinhibitory peptide (MIP) receptor. To test the functional role of SPR in mediating postmating responses in a non-Drosophila dipteran, we generated 2 independent Spr-knockout alleles in the yellow fever mosquito, Aedes aegypti. Although SPR is needed for postmating responses in Drosophila and the cotton bollworm Helicoverpa armigera, Spr mutant Ae. aegypti show completely normal postmating decreases in remating propensity and increases in egg laying. In addition, injection of synthetic SP or accessory gland homogenate from D. melanogaster into virgin female mosquitoes did not elicit these postmating responses. Our results demonstrate that Spr is not required for these canonical postmating responses in Ae. aegypti, indicating that other, as yet unknown, signaling pathways are likely responsible for these behavioral switches in this disease vector.

A semiochemical view of the ecology of the seed beetle Acanthoscelides obtectus Say (Coleoptera: Chrysomelidae, Bruchinae).

The dried bean beetle, Acanthoscelides obtectus, is an economically important pest of stored legumes worldwide. Tracking the human-aided dispersion of its primary hosts, the Phaseolus vulgaris beans, it is now widespread in most bean-growing areas of the tropics and subtropics. In temperate regions where it can only occasionally overwinter in the field, A. obtectus proliferates in granaries, having multiple generations a year. Despite its negative impact on food production, no sensitive detection or monitoring tools exist, and the reduction of local populations still relies primarily on inorganic insecticides as fumigating agents. However, in the quest to produce more nutritious food more sustainably and healthily, the development of environmentally benign crop protection methods is vital against A. obtectus. For this, knowledge of the biology and chemistry of both the host plant and its herbivore will underpin the development of, among others, chemical ecology-based approaches to form an essential part of the toolkit of integrated bruchid management. We review the semiochemistry of the mate- and host-finding behaviour of A. obtectus and provide new information about the effect of seed chemistry on the sensory and behavioural ecology of host acceptance and larval development.

Spray-induced and nanocarrier-delivered gene silencing system targeting juvenile hormone receptor components: potential application as fertility inhibitors for Adelphocoris suturalis management.

BACKGROUND: Adelphocoris suturalis is a destructive pest that attacks > 270 plants, including cotton, maize, soybean, and fruit trees. Adelphocoris suturalis can cause tremendous crop losses when the density exceeds economic thresholds, but because it can be both phytophagous and zoophytophagous it can serve as a natural enemy of other pests when the density is below the economic threshold. Effective control of its population is beneficial for maximizing yield and profits. RNA interference (RNAi) has potential to be a viable alternative to conventional pesticide-based pest management, but the lack of efficient double-stranded RNA (dsRNA) delivery systems and candidate genes are currently limiting factors for field applications. RESULTS: In this study, RNAi of juvenile hormone (JH) receptor components methoprene-tolerant (Met)/Taiman (Tai) in Adelphocoris suturalis reduced fertility. Based on this reproductive role, we targeted Adelphocoris suturalis Met and Tai for knockdown by coupling nanomaterial-dsRNA complexes with a transdermal spray delivery system. Within 12 h of adult emergence, females were sprayed with star polycation (SPc)-dsRNA formulations and the RNAi effects were assessed over time. RNAi knockdown efficiencies of 39-58% were observed at 5 days post-treatment and abnormal ovarian development was apparent by 10 days post-treatment. CONCLUSION: Our results show that spray-induced and nanocarrier-delivered gene silencing (SI-NDGS) system targeting JH signal genes effectively impaired oviposition, thus developing a novel RNA fertility inhibitor to control Adelphocoris suturalis populations. These results give new perspective on pest management and suggest broad prospects for field applications. © 2024 Society of Chemical Industry.

Effects of soil on the development, survival, and oviposition of Culex quinquefasciatus (Diptera: Culicidae) mosquitoes.

BACKGROUND: Water quality is known to influence the development and survival of larval mosquitoes, which affects mosquito-borne pathogen transmission as a function of the number of mosquitoes that reach adulthood and blood feed. Although water properties are known to affect mosquito development, few studies have investigated the link among soil properties, water quality, and mosquito development. Given the large number of ground-breeding mosquito species, this linkage is a potentially important factor to consider in mosquito ecology. In this study, we explored the effects of different soils on multiple life history parameters of the ground-breeding mosquito species Culex quinquefasciatus (Diptera: Culicidae). METHODS: Cx. quinquefasciatus larvae were reared in water combined with different soil substrates (sandy, silt, or clay loam textures) at increasing soil to water volume ratios, with and without the addition of organic matter (fish food). Gravid mosquitoes were offered different soil-water extracts to investigate soil effects on oviposition preference. RESULTS: Without the addition of organic matter, larval survival and development differed significantly among waters with different soil textures and volumes of substrate. Mosquitoes in water with clay loam soil survived longer and developed further than mosquitoes in other soil waters. Larvae survived for longer periods of time with increased volumes of soil substrate. Adding organic matter reduced the differences in larval survival time, development, and pupation among soil-water extracts. Adult female mosquitoes oviposited more frequently in water with clay loam soil, but the addition of organic matter reduced the soil effects on oviposition preference. CONCLUSIONS: This study suggests soil composition affects larval mosquito survival and development, as well as the oviposition preference of gravid females. Future studies could differentiate abiotic and biotic soil features that affect mosquitoes and incorporate soil variation at the landscape scale into models to predict mosquito population dynamics and mosquito-borne pathogen transmission.

A potential link between aromatics-induced oviposition repellency behaviors and specific odorant receptor of Aedes albopictus.

BACKGROUND: The Asian tiger mosquito, Aedes albopictus, is a competent vector for the spread of several viral arboviruses including dengue, chikungunya, and Zika. Several vital mosquito behaviors linked to survival and reproduction are primarily dependent on a sophisticated olfactory system for semiochemical perception. However, a limited number of studies has hampered our understanding of the relationship between the A. albopictus acute olfactory system and the complex chemical world. RESULTS: Here, we performed a qRT-PCR assay on antennae from A. albopictus of differing sex, age and physiological states, and found that AalbOr10 was enriched in blood-fed female mosquitoes. We then undertook single sensillum recording to de-orphan AalbOr10 using a panel of physiologically and behaviorally relevant odorants in a Drosophila 'empty neuron' system. The results indicated that AalbOr10 was activated by seven aromatic compounds, all of which hampered egg-laying in blood-fed mosquitoes. Furthermore, using a post-RNA interference oviposition assay, we found that reducing the transcript level of AalbOr10 affected repellent activity mediated by 2-ethylphenol at low concentrations (10-4 vol/vol). Computational modeling and molecular docking studies suggested that hydrogen bonds to Y68 and Y150 mediated the interaction of 2-ethylphenol with AalbOr10. CONCLUSION: We reveal a potential link between aromatics-induced oviposition repellency behaviors and a specific odorant receptor in A. albopictus. Our findings provide a foundation for identifying active semiochemicals for the monitoring or controlling of mosquito populations. © 2024 Society of Chemical Industry.

Orco mediates olfactory behavior and oviposition in the whitefly Bemisia tabaci.

Chemical signals play a central role in mediating insect feeding and reproductive behavior, and serve as the primary drivers of the insect-plant interactions. The detection of chemical signals, particularly host plant volatiles, relies heavily on the insect's complex olfactory system. The Bemisia tabaci cryptic species complex is a group of globally important whitefly pests of agricultural and ornamental crops that have a wide range of host plants, but the molecular mechanism of their host plant recognition is not yet clear. In this study, the odorant coreceptor gene of the Whitefly MEAM1 cryptic species (BtOrco) was cloned. The coding sequence of BtOrco was 1413 bp in length, with seven transmembrane structural domains, and it was expressed primarily in the heads of both male and female adult whiteflies, rather than in other tissues. Knockdown of BtOrco using transgenic plant-mediated RNAi technology significantly inhibited the foraging behavior of whiteflies. This inhibition was manifested as a reduced percentage of whiteflies responding to the host plant and a prolonged foraging period. Moreover, there was a substantial suppression of egg-laying activity among adult female whiteflies. These results indicate that BtOrco has the potential to be used as a target for the design of novel active compounds for the development of environmentally friendly whitefly control strategies.

An Odorant Receptor Expressed in Both Antennae and Ovipositors Regulates Benzothiazole-Induced Oviposition Behavior in Bactrocera dorsalis.

The oriental fruit fly,Bactrocera dorsalis (Hendel), is a notorious pest of fruit crops, causing severe damage to fleshy fruits during oviposition and larval feeding. Gravid females locate suitable oviposition sites by detecting the host volatiles. Here, the oviposition preference of antenna-removed females and the electrophysiological response of ovipositors to benzothiazole indicated that both antennae and ovipositors are involved in perceiving benzothiazole. Subsequently, odorant receptors (ORs) expressed in both antennae and ovipositors were screened, and BdorOR43a-1 was further identified to respond to benzothiazole using voltage-clamp recording. Furthermore, BdorOR43a-1-/- mutants were obtained using the CRISPR/Cas9 system and their oviposition preference to benzothiazole was found to be significantly altered compared to WT females, suggesting that BdorOR43a-1 is one of the important ORs for benzothiazole perception. Our results not only demonstrate the important role of antennae and ovipositors in benzothiazole-induced oviposition but also elucidate on the OR responsible for benzothiazole perception in B. dorsalis.

Host plant preference of Lygus hesperus (Hemiptera: Miridae) in 4 field crops: potato, alfalfa, carrot, and pea.

The western tarnished plant bug, Lygus hesperus (Knight), has emerged as a pest of potatoes (Solanum tuberosum L.) in the Lower Columbia Basin of Oregon and Washington. This species is generally found infesting several other field-grown crops in the region; however, their host preference is poorly understood. Thus, greenhouse cage experiments were conducted to evaluate L. hesperus host preference by simultaneously presenting adults with 4 host plants: potato, alfalfa, Medicago sativa L., carrot, Daucus carota L., and pea, Pisum sativum L. In addition, an oviposition test was conducted. The results indicated that L. hesperus actively chose as a host and as an oviposition substrate among the 4 host plants. We found a significantly higher number of adults on alfalfa and potato plants over carrot or pea plants at 6 h, 24 h, and 48 h after adults were released into the cage. However, 96 h after release, more L. hesperus were found in alfalfa. In addition, female L. hesperus strongly preferred potato and alfalfa plants as an oviposition substrate over carrot and pea plants at 96 h after release.