Comparative evaluation of two lures to attract female mosquitoes in an urban natural reserve during daytime.

The most widely used attractant to capture adult female mosquitoes is CO2. However, there are also baits available on the market that emit a scent resembling human skin. These baits were specifically designed to attract highly anthropophilic species such as Aedes albopictus and Aedes aegypti. In this study, we compare the effectiveness of CDC traps baited either with CO2 or with a commercial blend simulating skin odor, BG-Sweetscent, for trapping female mosquitoes during daylight hours in an urban reserve in the City of Buenos Aires. We employed a hurdle generalized linear mixed model to analyze trap capture probability and the number of mosquitoes captured per hour, considering the effects of attractant, mosquito species, and their interaction. Traps baited with CO2 captured ten mosquito species, while those baited with BG-Sweetscent captured six in overall significantly lower abundance. The odds of capturing mosquitoes were 292% higher for the CO2-baited traps than for those baited with BG-Sweetscent. No evidence of a combined effect of attractant type and species on female mosquito captures per hour was found. Results indicated that CDC traps baited with CO2 were more effective than those baited with BG-Sweetscent in capturing more mosquito species and a higher number of mosquitoes within each species, even if the species captured with CO2 exhibited a certain level of anthropophilia. This result has practical implications for mosquito surveillance and control in urban natural reserves.

Allelopathy and potential allelochemicals of Ligularia sagitta as an invasive plant.

Allelopathy is the main chemical means in the invasion process of exotic plants and one of the key factors in grassland degradation. In this experiment, we investigated the effects of ethyl acetate phase extract (EAE), n-butanol phase extract (BE) and aqueous phase extract (AE) from the aboveground (stems and leaves) and roots of Ligularia sagitta on seed germination and seedling growth of four Gramineae forages (Poa pratensis L. Festuca ovina L. Elymus nutans Griseb. Agropyron cristatum (L.) Gaertn.) in their sympatric domains and one Legosuminae forage (Medicago sativa L.). The chemical components in each phase extract of L. sagitta were determined with UHPLC-MS/MS non-targeted metabolomics, and the differential compounds were screened using Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA). Within a set concentration range, EAE significantly inhibited seed germination and seedling growth of four Gramineae forages. BE and AE acted mainly in the seedling growth stage and did not significantly inhibit forage seed germination. P. pratensis was most sensitive to L. sagitta extracts; at 2.0 mg/mL of EAE from roots, germination energy and germination rate of P. pratensis seeds were 0. L. sagitta extracts inhibited the growth of M. sativa seedlings and did not inhibit its seed germination. A total of 904 compounds were identified with UHPLC-MS/MS, among which 31, 64, 81 and 66 metabolites displayed different accumulation patterns in the four comparison groups (R.EAE vs. R.BE, R.EAE vs. R.AE, SL.EAE vs. SL.BE, SL.EAE vs. SL.AE), respectively. In particular, 9 compounds were found to be common up-regulated differential metabolites in the four comparison groups and were enriched in EAE. Additionally, N,N-dimethylaniline, Caffeic acid, 4-Hydroxybenzoic acid, 4-Hydroxybenzaldehyde and cis-9-Octadecenoic acid as potential allelochemicals in L. sagitta. The results of this study support efforts at finding alternative control plants for the restoration of poisonous grass-type degraded grasslands.

Allelochemicals of Alexandrium tamarense and its algicidal mechanism for Prorocentrum donghaiense and Heterosigma akashiwo.

The effects of culture filtrate of Alexandrium tamarense on Prorocentrum donghaiense and Heterosigma akashiwo were investigated, including determination of algal density, photosynthesis, intracellular enzyme content and activity. The filtrate of A. tamarense had a stronger inhibitory effect on P. donghaiense than H. akashiwo, and the inhibitory effect decreased with higher temperature treatment of the filtrate. Instantaneous fluorescence (Ft) and maximum quantum yield of photosystem II (Fv/Fm) values of both kinds of target algae were reduced as exposed to the filtrate of A. tamarense, which proved that allelopathy could inhibit the normal operation of photosynthetic system. The increase of Malondialdehyde (MDA) content of the two kinds of target algae indicated that the cell membrane was seriously damaged by allelochemicals released by A. tamarense. The different responses of Superoxide Dismutase (SOD) and Catalase (CAT) activity in two kinds of target algae demonstrated the complexity and diversity of allelopathic mechanism. The filtrate of A. tamarense also influenced the metabolic function (ATPases) of P. donghaiense and H. akashiwo, and the influence on P. donghaiense was greater. Liquid-liquid extraction was used to extract and isolate allelochemicals from the filtrate of A. tamarense. It was found that only component I with molecular weight of 424.2573 and 434.2857 could inhibit the growth of P. donghaiense by HPLC-MS.

Chemical symphony: Unraveling triatomine aggregation and attraction patterns for innovative pest control solutions.

Aggregation is a spatial distribution pattern where individuals can be grouped through interaction with particular signals or cues [e.g., chemical substances]. Numerous triatomine species exhibit attraction and aggregation around both conspecific and heterospecific feces. It remains unclear whether compounds released by feces function as signals (pheromones or synomones) or cues. Employing a bioassay that mimics field conditions, we investigated the response of Triatoma pallidipennis bugs to volatiles present in its feces and blends of these compounds. Our findings indicate that short-chain aldehydes, namely nonanal, octanal, heptanal, and hexanal, attract fifth-instar nymphs. Furthermore, insects responded to individual compounds as well as secondary, ternary, and quaternary blends. The most attractive blend consisted of all four aldehydes. Additionally, quaternary blends at various compound ratios attracted fifth-instar nymphs of T. phyllosoma and T. longipennis. We discuss the potential roles of these compounds as signals or cues and explore their application as bait in control programs.

Termites can learn.

It is generally believed that termites can't learn and are not "intelligent". This study aimed to test whether termites could have any form of memory. A Y-shaped test device with one release chamber and two identical test chambers was designed and constructed by 3D printing. A colony of damp wood termites was harvested from the wild. Worker termites were randomly selected for experiment. Repellent odors that could mimic the alarm pheromone for termites were first identified. Among all substances tested, a tea tree oil and lemon juice were found to contain repellent odors for the tested termites, as they significantly reduced the time that termites spent in the chamber treated with these substances. As control, a trail pheromone was found to be attractive. Subsequently, a second cohort of termites were operant conditioned by punishment using both tea tree oil and lemon juice, and then tested for their ability to remember the path that could lead to the repellant odors. The test device was thoroughly cleaned between trials. It was found that conditioned termites displayed a reduced tendency to choose the path that led to expectant punishment as compared with naïve termites. Thus, it is concluded that damp wood termites are capable of learning and forming "fear memory", indicative of "intelligence" in termites. This result challenges established presumption about termites' intelligence.

Allelochemicals-mediated interaction between algae and bacteria: Direct and indirect contact.

Secondary metabolites with bioactivity are allelochemicals. This study adopted direct contact (R0) and indirect contact (separated by 0.45 µm membrane, R1-A for algae, R1-S for sludge) to reveal the role of metabolites especially allelochemicals on interaction of bacteria and algae. Direct contact exhibited better nutrients removal than indirect contact, due to less antibacterial allelochemicals and oxidative stress. Bacterial signaling molecules were not detected. The major algae-derived allelochemicals were 13-Docosenamide, 9-Octadecenamide, n-Hexadecanoic acid, erucic acid, octadecanoic acid, ß-sitosterol, and E,E,Z-1,3,12-Nonadecatriene-5,14-diol. Furthermore, presence of 13-Docosenamide and 9-Octadecenamide was associated with succession of Flavobacterium and suppression of nitrifying bacteria (Nitrosomonas, Ellin6067, and Nitrospira). Direct contact stimulated denitrifying bacteria Saccharimonadales and algae Scenedesmus, whereas indirect contact is friendly to Dechloromonas, Competibacter, nitrifying bacteria, algae Desmodesmus and Dictyosphaerium. This study highlights the essentiality of cell contact of bacteria-algae in establishing synergy, as cell contact mitigates antagonistic effect induced by metabolites.

Differential interference effects of thermal pollution on the induced defense of different body-sized cladocerans.

Climate warming influences the biological activities of aquatic organisms, including feeding, growth, and reproduction, thereby affecting predator-prey interactions. This study explored the variation in thermal sensitivity of anti-predator responses in two cladoceran species with varying body sizes, Daphnia pulex and Ceriodaphnia cornuta. These species were cultured with or without the fish (Rhodeus ocellatus) kairomone at temperatures of 15, 20, 25, and 30 °C for 15 days. Results revealed that cladocerans of different body sizes exhibited varying responses to fish kairomones in aspects such as individual size, first-brood neonate size, total offspring number, average brood size, growth rate, and reproductive effort. Notably, low temperature differently affected defense responses in cladocerans of different body sizes. Both high and low temperatures moderated the intensity of the kairomone-induced response on body size at maturity. Additionally, low temperature reversed the reducing effect of fish kairomone on the total offspring number, average brood size, and reproductive effort in D. pulex. Conversely, it enhanced the increasing effect of fish kairomone on these parameters in C. cornuta. These results suggest that inducible anti-predator responses in cladocerans are modifiable by temperature. The differential effects of fish kairomones on various cladocerans under temperature influence offer crucial insights for predicting changes in predator-prey interactions within freshwater ecosystems under future climate conditions.

(R)-(+)-γ-Decalactone is Conserved in North America as a Pheromone Component of Osmoderma eremicola (Coleoptera: Scarabaeidae) and a Kairomone of Elater abruptus (Coleoptera: Elateridae).

The scarab genus Osmoderma (Coleoptera: Scarabaeidae) includes several large species called hermit beetles that develop within dead and decaying hardwood trees. Males of at least three Palearctic species produce the aggregation-sex pheromone (R)-(+)-γ-decalactone, including the endangered O. eremita (Scopoli). However, hermit beetles have received less attention in the western hemisphere, resulting in a large gap in our knowledge of the chemical ecology of Nearctic species. Here, we identify (R)-( +)-γ-decalactone as the primary component of the aggregation-sex pheromone of the North American species Osmoderma eremicola (Knoch). Field trials at sites in Wisconsin and Illinois revealed that both sexes were attracted to lures containing (R)-(+)-γ-decalactone or the racemate, but only males of O. eremicola produced the pheromone in laboratory bioassays, alongside an occasional trace of the chain-length analog γ-dodecalactone. Females of the congener O. scabra (Palisot de Beauvois) were also significantly attracted by γ-decalactone, suggesting further conservation of the pheromone, as were females of the click beetle Elater abruptus Say (Coleoptera: Elateridae), suggesting that this compound may have widespread kairomonal activity. Further research is needed to explore the behavioral roles of both lactones in mediating behavioral and ecological interactions among these beetle species.

Enhancing entomopathogenic nematode efficacy with Pheromones: A field study targeting the pecan weevil.

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

Protocol for monitoring and analyzing pheromone-mediated behavioral response of sea lamprey in a natural system.

Olfactory-mediated behaviors in fish are often examined in artificial microcosms that enable well-controlled treatments but fail to replicate environmental and social contexts. However, observing these behaviors in nature poses challenges. Here, we describe a protocol for recording sea lamprey (Petromyzon marinus) behaviors in a natural system. We describe steps for administering and verifying accurate odorant concentrations, surveying sea lamprey abundance, and tracking sea lamprey movements. We also detail procedures to analyze treatment effects on pheromone-mediated spawning in a high-density population. For complete details on the use and execution of this protocol, please refer to Scott et al.1.

Discovery and properties of novel analogues of the aphid pheromones nepetalactone and nepetalactol.

BACKGROUND: Pheromones have unique advantages for pest control. Current aphid pheromone research focuses on alarm and sex pheromones. However, practical applications are limited so far, as (E)-ß-farnesene has only been investigated to a small extent as an alarm pheromone and only male aphids are targeted by sex pheromones. Previous literature reports electrophysiological responses and repellent behavior of asexual aphids to nepetalactone (1B), therefore our objective was to modify nepetalactone's structure to identify key fragments responsible for repellent effects, as guidance for subsequent modifications and further investigation. RESULTS: In this study, seven derivatives were designed and synthesized based on nepetalactol (1A) and nepetalactone (1B) as lead compounds. Free-choice tests, conducted using cowpea aphids (Aphis craccivora), revealed that the lactone moiety was crucial for the repellent activity, and the removal of the carbonyl group eliminated the repelling effect. Compound (±)1I, an analogue of nepetalactone (1B), demonstrated a significantly higher repellent value than nepetalactone (1B) at three different concentrations, and even at 0.1 mg/mL it maintained a considerable repellent effect (26.5%). Electrostatic potential and density functional theory calculations supported the importance of the carbonyl group for the repellent effects. CONCLUSION: The newly discovered para-pheromone (±)1I shows improved repellent effects and potential for development as a novel biological control agent. Based on our innovative findings, analogues with improved efficacy and properties can be designed and prepared. Our research contributes to understanding the effects of structural modifications on pheromone activity and properties, which is crucial for exploring novel pheromone-based products for crop protection. © 2024 Society of Chemical Industry.

Repellent activity of the non-host semiochemical (E)-2-octenal against Amblyomma sculptum and Amblyomma dubitatum ticks under field conditions.

Amblyomma ticks pose a significant public health threat due to their potential to transmit pathogens associated with rickettsial diseases. (E)-2-octenal, a compound found in donkeys (Equus asinus), exhibits strong repellent properties against Amblyomma sculptum nymphs under laboratory conditions. This study assessed the effectiveness of the (E)-2-octenal in wearable slow-release devices for personal human protection against Amblyomma ticks under natural conditions. Slow-release devices treated with (E)-2-octenal and untreated controls were prepared and tested on two volunteers walking through a tick-infested area in Goiania, Brazil. The experiment was conducted twice daily for three series of 10 days, with each volunteer wearing two devices attached to each leg, one on the ankle and one just above the thigh. Volunteers with control and treated devices exchanged them between rounds. Also, the daily release rate of (E)-2-octenal from the slow-release devices was determined in the laboratory, increasing significantly from 0.77 ± 0.14 µg/day on the first day to 9.93 ± 1.92 µg/day on the 4th day and remaining constant until the 16th day. A total of 5409 ticks were collected from both volunteers. Treated devices resulted in recovering fewer ticks (n = 1,666; 31%) compared to untreated devices (control: n = 3,743; 69%). (E)-2-octenal effectively repelled Amblyomma spp. larvae, A. sculptum adults, and exhibited pronounced repellency against A. dubitatum nymphs and adults. These findings suggest the potential of (E)-2-octenal delivered by wearable slow-release devices as a green-based repellent. Further improvements, however, are necessary to provide better protection for humans against A. sculptum and A. dubitatum in field conditions.

Developing attractants and repellents for ticks: promises and challenges.

Historically, some of the most effective tools to counter vector-borne diseases have been those directed against the vectors. Ticks are undergoing a population explosion as evidenced by the recent expansion of their distribution range. Tick control has traditionally relied heavily on pesticides. However, sustained use of acaricides is resulting in resistant tick populations. Multipronged management strategies that build and expand upon innovative control methods are sorely needed. Behavior-modifying chemicals, referred to as semiochemicals, such as pheromones and repellents, offer a first line of personal protection against ticks. We review the current understanding of tick semiochemicals, and how such understanding is leading to the identification of novel chemistries that are effective and safe.

Visual stimulus brightness influences the efficiency of attractant-baited traps for catching Drosophila suzukii Matsumura (Diptera: Drosophilidae).

Drosophila suzukii (Matsumura) is an exotic pest of economic importance that affects several soft-skinned fruits in Mexico. Previously, we found that yellow or yellow-green rectangular cards inside a transparent trap baited with attractants improved D. suzukii capture. In this study, we evaluated the influence of rectangular cards with different yellow shades inside a transparent multi-hole trap baited with apple cider vinegar (ACV) on D. suzukii capture in the field. Second, we tested whether ACV-baited traps with cards of other geometric shapes affected D. suzukii catches compared to traps with rectangular cards. Third, we evaluated the effects of commercial lures combined with a more efficient visual stimulus from previous experiments on trapping D. suzukii flies. We found that ACV-baited traps plus a yellow-shaded rectangle card with 67% reflectance at a 549.74 nm dominant wavelength captured more flies than ACV-baited traps with yellow rectangle cards with a higher reflectance. Overall, ACV-baited traps with rectangles and squares caught more flies than did ACV-baited traps without visual stimuli. The traps baited with SuzukiiLURE-Max, ACV and Z-Kinol plus yellow rectangles caught 57, 70 and 101% more flies, respectively, than the traps baited with the lure but without a visual stimulus.

Spectral sensitivity of click beetles (Coleoptera: Elateridae) and their responses to light stimuli in laboratory and field experiments.

With increasingly fewer insecticides registered to control the larvae of pest click beetles (Coleoptera: Elateridae), integrative beetle management, including pheromone- and light-based trapping of adult beetles, must be explored as an alternative strategy. Here, we analyzed the spectral sensitivity and color preference of 9 elaterids across 6 genera in electrophysiological recordings and in behavioral bioassays. In electroretinogram recordings (ERGs), dark-adapted beetles were exposed to narrow wavebands of light in 10-nm increments from 330 to 650 nm. All beetles proved most sensitive to green (515-538 nm) and ultraviolet (UV) light (~360 nm). In 4-choice bioassay arenas with 3 light emitting diodes (LEDs; green [525 nm], blue [470 nm], red [655 nm]) and a dark control as test stimuli, beetles discriminated between test stimuli, being preferentially attracted to green and blue LEDs. In field experiments, Vernon pitfall traps fitted with a green, blue or white LED captured significantly more male and female Agriotes lineatus and A. obscurus than dark control traps. When traps were baited with green or blue LEDs at light intensities that differed by 10-fold, the traps baited with higher light intensity lures captured numerically more beetles but trap catch data in accordance with light intensity did not differ statistically. Light-based trapping may be a viable tool for monitoring elaterid species known not to have pheromones.

Courtship and mating behavior of Endoclita signifer (Hepialidae: Lepidoptera) and the male sex pheromones in hairbrushes.

Endoclita signifer Walker is the most destructive wood-boring pest of Eucalyptus in China, causing significant economic and ecological damage. As an insect of the primitive Lepidoptera family Hepialidae, E. signifer fly and mat for only 10-20 min at dusk. The courtship and mating behavior of E. signifer adults and whether male moths release sex pheromones are still unknown, especially since transitory flight survival strategies in primitive moths differ from advanced moths like noctuids. In this study, we first observed the courtship and mating behavior of E. signifer by considering the effects of space and then analyzed extracts of male hairbrushes using gas chromatography-electroantennogram detection. Our results indicated that during the courtship period, flying males form courtship fields, lekking, and chase flying females before mating with them; E. signifer were more successful in mating in larger spaces (Length × Width × Height = 9.6 × 7 × 4 m); 5 compounds in the hairbrushes of the male moths which elicited antennal responses of 2 sexes, despite at high concentrations. Combined with it, indicating that communication between male and female may rely on male sex pheromones. These findings can serve as a basis for studying the mechanisms of sex communication in E. signifer and developing sex pheromone-based trapping techniques.

Disruption of semiochemical-mediated movement by the immature Trogoderma variabile Baillon and Trogoderma inclusum Le Conte (Coleoptera: Dermestidae) after exposure to long-lasting insecticide-incorporated netting.

BACKGROUND: Highly mobile stored product insects may be able to readily orient in response to food cues and pheromones to attack durable commodities at each link of the postharvest supply chain. A 0.4% deltamethrin-incorporated long-lasting insecticide-incorporated netting (LLIN) is a successful novel preventative integrated pest management (IPM) tactic to intercept dispersing insects after harvest. However, it is unknown whether exposure to LLIN may affect olfaction and orientation to important semiochemicals by immature stored product dermestids, therefore the aim of this study was to assess whether exposure to LLIN disrupts the normal olfactory and chemotactic behavior of warehouse beetle, Trogoderma variabile Ballion (Coleoptera: Dermestidae), and the larger cabinet beetle, T. inclusum Le Conte (Coleoptera: Dermestidae), larval movement in the presence of important semiochemicals, including food kairomones (e.g., flour) and pheromones, e.g., (Z)-14-methyl-8-hexadecenal. RESULTS: The distance moved by the larval population of T. variabile was reduced by 64% after 24-h exposure to LLIN compared to control netting but not immediately after exposure, while T. inclusum larvae movement was reduced by 50% after 24-h exposure to LLIN compared to the control netting. Generally, the olfaction and orientation of larval dermestids were affected after exposure to LLIN compared to control netting. There were species-linked differences in effects on olfaction after the insects were exposed to LLIN. CONCLUSION: Our study suggests the use of LLIN may enhance the effectiveness of other concurrent behaviorally-based strategies such as mating disruption when used as part of a comprehensive IPM program in the postharvest environment. © 2023 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Sequential Isolation of Essential Oils Repellent to the Red Palm Weevil Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae).

The push-pull approach using semiochemicals in pest control requires both an attractant and a repellent. Many previous studies have arbitrarily tested one or more known insect repellents or plant essential oils (EOs) hoping to find repellents of an insect pest. We used a comprehensive approach that synergistically tests in the field numerous natural volatiles from commercial EOs to identify repellents of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae), a worldwide pest of palms and date palms. Volatiles from 79 EOs in slow-release devices were divided into five groups and tested in traps with attractive pheromone compared to traps with pheromone alone. EO-treatment groups exhibiting repellency due to significant trap shutdown, were further subdivided into subgroups of four EOs each and tested further. Two groups of four EOs (cypress, desert wormwood, elemi, and Eucalyptus citriodora) and (niaouli, nutmeg, oregano, and orange sweet), or their corresponding mixtures of major volatiles, caused pheromone trap reductions of up to 92%. Further tests showed that seven of the eight EOs are similarly repellent as the corresponding subgroup. This systematic approach of successively testing sub-fractions of EOs in the field for trap shutdown should be useful to identify repellents of other insect pests of crops.

Allelochemicals: A source for developing economically and environmentally friendly plant growth regulators.

Allelochemicals are specific secondary metabolites that can exhibit autotoxicity by inhibiting the growth of the same plant species that produced them. These metabolites have been found to affect various physical processes during plant growth and development, including inhibition of seed germination, photosynthesis, respiration, root growth, and nutrient uptake, with diverse mechanisms involving cell destruction, oxidative homeostasis and photoinhibition. In some cases, allelochemicals can also have positive effects on plant growth and development. In addition to their ecological significance, allelochemicals also possess potential as plant growth regulators (PGRs) due to their extensive physiological effects. However, a comprehensive summary of the development and applications of allelochemicals as PGRs is currently lacking. In this review, we present an overview of the sources and categories of allelochemicals, discuss their effects and the underlying mechanisms on plant growth and development. We showcase numerous instances of key phytohormonal allelochemicals and non-phytohormonal allelochemicals, highlighting their potential as candidates for the development of PGRs. This review aims to provide a theoretical basis for the development of economical, safe and effective PGRs utilizing allelochemicals, and emphasizes the need for further research in this area.

Allelochemical root-growth inhibitors in low-molecular-weight cress-seed exudate.

BACKGROUND AND AIMS: Cress seeds release allelochemicals that over-stimulate the elongation of hypocotyls of neighbouring (potentially competing) seedlings and inhibit their root growth. The hypocotyl promoter is potassium, but the root inhibitor was unidentified; its nature is investigated here. METHODS: Low-molecular-weight cress-seed exudate (LCSE) from imbibed Lepidium sativum seeds was fractionated by phase partitioning, paper chromatography, high-voltage electrophoresis and gel-permeation chromatography (on Bio-Gel P-2). Fractions, compared with pure potassium salts, were bioassayed for effects on Amaranthus caudatus seedling growth in the dark for 4 days. KEY RESULTS: The LCSE robustly promoted amaranth hypocotyl elongation and inhibited root growth. The hypocotyl inhibitor was non-volatile, hot acid stable, hydrophilic and resistant to incineration, as expected for K+. The root inhibitor(s) had similar properties but were organic (activity lost on incineration). The root inhibitor(s) remained in the aqueous phase (at pH 2.0, 6.5 and 9.0) when partitioned against butan-1-ol or toluene, and were thus hydrophilic. Activity was diminished after electrophoresis, but the remaining root inhibitors were neutral. They became undetectable after paper chromatography; therefore, they probably comprised multiple compounds, which separated from each other, in part, during fractionation. On gel-permeation chromatography, the root inhibitor co-eluted with hexoses. CONCLUSIONS: Cress-seed allelochemicals inhibiting root growth are different from the agent (K+) that over-stimulates hypocotyl elongation and the former probably comprise a mixture of small, non-volatile, hydrophilic, organic substances. Abundant components identified chromatographically and by electrophoresis in cress-seed exudate fitting this description include glucose, fructose, sucrose and galacturonic acid. However, none of these sugars co-chromatographed and co-electrophoresed with the root-inhibitory principle of LCSE, and none of them (in pure form at naturally occurring concentrations) inhibited root growth. We conclude that the root-inhibiting allelochemicals of cress-seed exudate remain unidentified.